Titanium sheet / MXene composite material and preparation method and application thereof

Abstract

The invention discloses a titanium sheet / MXene composite material and a preparation method and application thereof, and relates to the technical field of medical materials.The preparation method comprises the following steps that 1, titanium aluminum carbide powder is subjected to acid etching, centrifugation, washing, ultrasonic stripping and drying, and MXene nanosheets are obtained; (2) dispersing the MXene nanosheets in deionized water, so as to obtain an MXene dispersion liquid; and (3) coating the surface of the titanium sheet subjected to alkali etching with the MXene dispersion liquid, and drying to obtain the titanium sheet / MXene composite material. The composite material can inhibit expression of macrophage inflammatory factors and promote M2 polarization of macrophages, so that inflammation is relieved, and the treatment potential of the composite material in the aspect of medical implant application is highlighted through a unique mechanism of promoting osteoblast differentiation and vascular cell differentiation through autophagy mediation.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a titanium sheet / MXene composite material, its preparation method, and its applications. Background Technology

[0002] Joint replacement surgery, which replaces damaged autologous bone tissue with prostheses, is primarily used to treat end-stage osteoarthritis and other bone and joint diseases. Titanium and its alloys are widely used in orthopedic and dental implants due to their excellent biocompatibility, high mechanical strength, and superior corrosion resistance. With continuous advancements in surgical techniques and manufacturing processes, global demand for artificial joint replacements (including knee, hip, and temporomandibular joint prostheses) is steadily increasing. However, the bioinertness of titanium implants limits their interaction with surrounding biological tissues, leading to poor osseointegration, delayed healing, and even implant failure in some cases. Therefore, there is an urgent need to develop novel strategies to enhance the osseointegration capacity of titanium implants.

[0003] To overcome these limitations, researchers have developed various surface modification strategies to enhance the biocompatibility of titanium implants. Physicochemical treatments (such as sandblasting, acid-base etching, anodizing, and sol-gel deposition) can alter surface roughness and chemical composition, thereby enhancing protein adsorption, cell adhesion, and osteogenic differentiation. Furthermore, the spatial and physical design of the implant surface can modulate immune responses by controlling macrophage morphology and behavior. Notably, micro-roughened titanium surfaces with superhydrophilic properties have been shown to promote macrophage polarization towards the anti-inflammatory M2 phenotype, thereby promoting the secretion of osteogenic growth factors that support osteoblast differentiation. Among these methods, alkaline etching has attracted considerable attention due to its ease of operation and ability to create superhydrophobic surfaces with nanoscale roughness. This nanostructure topography is highly similar to the hierarchical structure of natural bone and plays a crucial role in guiding cellular responses at the bone-implant interface. However, simply increasing implant surface roughness is insufficient to address key challenges such as chronic inflammation, which significantly hinder healing and jeopardize the long-term stability of the implant.

[0004] In recent years, two-dimensional nanomaterials, especially those with the molecular formula M n+1 X n T x Transition metal carbides and nitrides (MXenes) have shown great potential as surface coatings in the biomedical field. MXenes stand out due to their unique mechanical, thermal, optical, chemical, and electronic properties, offering broad prospects for various applications. Recent research indicates that MXenes demonstrate significant potential in bone tissue engineering and are expected to become ideal candidate materials for implant surface modification. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a titanium sheet / MXene composite material. First, an alkaline etching process is used to form a biomimetic rough surface on a titanium substrate, and then MXene nanosheets are deposited on its surface. This dual modification method aims to synergistically combine the micro-topographic advantages of etched titanium with the functional properties of MXene to achieve dual optimization of surface structure and bioactivity. The final composite material has excellent anti-inflammatory and osteointegration properties.

[0006] The technical problem to be solved by this invention is achieved by the following technical solution: The first objective of this invention is to provide a method for preparing a titanium sheet / MXene composite material, comprising the following steps: (1) Acid etching, centrifugation, washing, ultrasonic peeling and drying of titanium aluminum carbide powder were performed to obtain MXene nanosheets; (2) MXene nanosheets were dispersed in deionized water to obtain an MXene dispersion; (3) The MXene dispersion was coated onto the surface of the alkali-etched titanium sheet and dried to obtain the titanium sheet / MXene composite material.

[0007] Furthermore, the acid etching uses a LiF / HCl mixed solution as the etching solution, wherein the molar ratio of LiF to HCl is 1:(2~3).

[0008] Furthermore, the ratio of the titanium aluminum carbide powder to the LiF / HCl mixed solution is 1 g : (10~20) mL.

[0009] Furthermore, the acid etching temperature is 30~40℃, and the time is 24~28h.

[0010] Furthermore, the washing is a water wash, the purpose of which is to remove unreacted MAX phase, byproducts (aluminum fluoride), and residual etching agent. Multiple washes yield a high-purity, neutral pH MXene suspension, avoiding acidic or salt residues that could lead to MXene oxidation or performance degradation.

[0011] Furthermore, the ultrasonic ablation frequency is 20~40 kHz, the power is 100~500 W, and the time is 0.5~2 h.

[0012] Furthermore, the concentration of the MXene dispersion is 0.025~10 mg / mL.

[0013] Furthermore, the alkaline etching uses potassium hydroxide solution as the etching solution with a concentration of 1~5 M.

[0014] Furthermore, the alkaline etching temperature is 30~50℃, and the time is 1~8h.

[0015] Furthermore, the coating method includes, but is not limited to, at least one of drop coating, roller coating, brush coating, spray coating, and dip coating. The coating thickness is determined according to the concentration of the MXene dispersion, so that a certain amount of MXene nanosheets are coated on the surface of the titanium sheet.

[0016] A second objective of this invention is to provide a titanium sheet / MXene composite material prepared by the aforementioned preparation method.

[0017] A third objective of the present invention is to provide the use of the titanium sheet / MXene composite material as a medical implant.

[0018] The beneficial effects of this invention are as follows: This invention prepares an alkali-etched titanium sheet / MXene composite material by alkali etching titanium sheet surface composite with MXene. This composite material can inhibit the expression of macrophage inflammatory factors, promote macrophage M2 polarization, thereby reducing inflammation, and promote osteoblast differentiation and angiogenesis differentiation through a unique mechanism mediated by autophagy, highlighting its therapeutic potential in medical implant applications. Attached Figure Description

[0019] Figure 1 The effects of alkaline-etched titanium sheets and un-alkaline-etched titanium sheets prepared in Examples 1-4 on the viability of bone marrow mesenchymal stem cells (BMSCs), human umbilical vein endothelial cells (HUVECs), and mouse macrophages (RAW264.7). Figure 2 Effects of TA and AE-MXene prepared in Examples 1, 5-9 and AE prepared in Comparative Example 2 on the viability of BMSCs, HUVECs and RAW264.7 cells; Figure 3 Optical images of AE-MXene prepared for TA and Examples 1, 5-9, and AE prepared for Comparative Example 2; Figure 4 The staining results of AE-MXene prepared for TA and Examples 1 and 7, and AE prepared for Comparative Example 2 on BMSCs, HUVECs and RAW264.7 cells; Figure 5 The results of the Zeta potential test for the MXene dispersions prepared in Examples 1, 5-7 are shown. Figure 6 The results of dynamic light scattering (DLS) particle size analysis of the MXene dispersions prepared in Examples 1, 5-7 are shown. Figure 7 Scanning electron microscope (SEM) images of TA, AE-MXene prepared in Example 1, TA-MXene prepared in Comparative Example 1, and AE prepared in Comparative Example 2. Figure 8X-ray diffraction (XRD) patterns of TA and TA-MXene prepared in Comparative Example 1; Figure 9 X-ray photoelectron spectroscopy (XPS) spectra of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2; Figure 10 An atomic force microscope (AFM) image of AE-MXene prepared in Example 1; Figure 11 Roughness test results for TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2; Figure 12 The water contact angle test results of TA, AE-MXene prepared in Example 1 and AE prepared in Comparative Example 2; Figure 13 The effects of TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2 on the mRNA expression of pro-inflammatory cytokines; Figure 14 To analyze the effects of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2 on the expression of M1 marker (CD86) and M2 marker (CD206) genes in RAW264.7 cells under inflammatory conditions by flow cytometry; Figure 15 To analyze the effects of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2 on the expression of M1 markers (Il11, Il18, Il23a) and M2 markers (Mrc1, Arg1, Pparg, Retnla) in RAW264.7 cells under inflammatory conditions using RT-qPCR. Figure 16 The effects of TA and AE-MXene prepared in Examples 1, 5-7 and AE prepared in Comparative Example 2 on alkaline phosphatase (ALP) activity in BMSCs cells; Figure 17 To analyze the effects of TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2 on the expression of osteogenic genes (Runx2, Bglap, Col1a1, Sp7, and Alpl) in BMSCs cells by RT-qPCR; Figure 18 Immunofluorescence images of HUVECs cells treated with TA and AE-MXene prepared in Examples 1, 6, and 7, respectively, and AE prepared in Comparative Example 2. Figure 19 To perform quantitative analysis using ImageJ software Figure 18 The network structure and number of branches in it; Figure 20To use RT-qPCR analysis Figure 18 Expression of VEGF and ANG in HUVECs cells; Figure 21 A flowchart for mass spectrometry sequencing of RAW264.7 cells cultured on TA and AE-MXene; Figure 22 for Figure 21 Figure showing the results of functional enrichment analysis by mid-mass spectrometry sequencing; Figure 23 Representative images and corresponding quantitative statistical analysis graphs of autophagic flux visualization detection using the mRFP-GFP-LC3 combined fluorescence reporter system, along with the number of APs, ALs, and the AL / AP ratio. Figure 24 A schematic diagram illustrating the acquisition and use of macrophage culture medium (CM); Figure 25 The results of ALP staining and Alizarin Red S staining of BMSCs cells after CM treatment; Figure 26 The results of immunofluorescence staining for osteogenic markers in BMSCs cells after CM treatment; Figure 27 Immunofluorescence images of HUVECs cells after CM treatment and quantitative analysis of the network structure and branch number in the immunofluorescence images; Figure 28 To analyze the expression of VEGF and ANG in HUVECs cells treated with CM using RT-qPCR. Detailed Implementation

[0020] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments and illustrations.

[0021] The following examples and comparative examples use abbreviations for explanation: Titanium sheet, abbreviated as TA; Alkali etched titanium sheet, abbreviated as AE; Alkali-etched titanium sheet / MXene composite material, abbreviated as AE-MXene; Titanium sheet / MXene composite material, abbreviated as TA-MXene.

[0022] The titanium aluminum carbide powder used in the embodiments and comparative examples of this invention was purchased from Ningbo Jinlei Nanomaterials Technology Co., Ltd., with a particle size of 400 mesh and a purity of 99%; the titanium sheet was purchased from Hefei Wenghe Metal Materials Co., Ltd., with a thickness of 0.2 mm. The pretreatment method included: first polishing the titanium sheet with metallographic sandpaper, then cleaning it with acetone, ethanol and deionized water, and then drying it with nitrogen gas flow.

[0023] The cell experiments of this invention were approved by the Animal Nursing and Experimentation Committee of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SH9H-2024-A1434-1). Primary BMSCs were suspended in MEM-α medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Third-generation BMSCs were used in the experiments. HUVECs were purchased from OriCell (Shanghai, China) and cultured in angiogenesis induction medium and Matrigel. Both the RAW 264.7 and THP-1 cell lines were derived from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RAW 264.7 cells were cultured in DMEM medium containing 5% fetal bovine serum and 1% penicillin / streptomycin, and THP-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0024] Synthesis and structural characterization: Example 1 S1. Add 2 g of titanium aluminum carbide powder to 30 mL of LiF / HCl mixed solution (the molar ratio of LiF to HCl is 1:2.94), acid etch at 40℃ for 48 h, centrifuge, wash with water until pH value is 6-7, purge the resulting suspension with nitrogen for 10 min, then ultrasonically exfoliate at a frequency of 40 kHz and a power of 200 W for 1 h, freeze dry to obtain MXene nanosheets.

[0025] S2. MXene nanosheets were ultrasonically dispersed in water at a frequency of 40 kHz, a power of 200 W, and a time of 15 min to obtain an MXene dispersion with a concentration of 0.1 mg / mL.

[0026] S3. Immerse the titanium sheet in a 4 M potassium hydroxide solution and etch it at 50 °C for 2 h. Centrifuge, wash with water, and dry to obtain the alkali-etched titanium sheet.

[0027] S4. The MXene dispersion was uniformly drop-coated onto the surface of the alkaline-etched titanium sheet with a coating thickness of 10 μm. The coating was then dried at 50°C for 4 h to obtain the alkaline-etched titanium sheet / MXene composite material.

[0028] Examples 2-4 The titanium sheet was alkali etched according to step S3 in Example 1, except that the alkali etching time was adjusted to 1h, 4h, and 8h respectively, and the alkali etched titanium sheets obtained by step S3 in Examples 2, 1, 3, and 4 were named AE-1h, AE-2h, AE-4h, and AE-8h respectively.

[0029] The toxicity of alkaline-etched titanium sheets and unalkaline-etched titanium sheets prepared in Examples 1-4 to bone marrow mesenchymal stem cells (BMSCs), human umbilical vein endothelial cells (HUVECs), and mouse macrophages (RAW264.7) was determined using a CCK-8 assay kit. BMSCs, HUVECs, and RAW264.7 cells were seeded onto alkaline-etched and unalkaline-etched titanium sheets prepared in Examples 1-4, respectively, and cultured in a 37°C, 5% CO2 incubator. Then, 1 / 10 of the culture medium volume of CCK-8 reagent was added to each well, and the cells were cultured for another 4 h. The absorbance was measured at 450 nm, and cell viability was calculated. Figure 1 As shown. By Figure 1 It can be seen that when the alkaline etching time is 2 h, the alkaline etching of titanium sheets has the best effect on promoting the proliferation of these three types of cells.

[0030] Examples 5-9 Alkali-etched titanium / MXene composite materials were prepared according to the method in Example 1, except that the concentration of the MXene dispersion was adjusted to 0.025 mg / mL, 0.05 mg / mL, 0.2 mg / mL, 1 mg / mL, and 10 mg / mL, respectively.

[0031] Comparative Example 1 (1) 2 g of titanium aluminum carbide powder was added to 30 mL of LiF / HCl mixed solution (the molar ratio of LiF to HCl was 1:2.94), acid etched at 40℃ for 48 h, centrifuged, washed with water until pH value was 6-7, nitrogen gas was introduced into the resulting suspension for 10 min, and then ultrasonically exfoliated at a frequency of 40 kHz and a power of 200 W for 1 h, and freeze-dried to obtain MXene nanosheets.

[0032] (2) MXene nanosheets were ultrasonically dispersed in water at a frequency of 40 kHz, a power of 200 W, and a time of 15 min to obtain an MXene dispersion with a concentration of 0.1 mg / mL.

[0033] (3) The MXene dispersion was uniformly drop-coated onto the surface of the titanium sheet with a coating thickness of 10 μm and dried at 50℃ for 4 h to obtain the titanium sheet / MXene composite material.

[0034] Comparative Example 2 The titanium sheet was immersed in a 4 M potassium hydroxide solution and etched at 50 °C for 2 h. After centrifugation, washing with water, and drying, the alkali-etched titanium sheet was obtained.

[0035] The toxicity of TA, AE-MXene prepared in Examples 1, 5-9, and AE prepared in Comparative Example 2 to BMSCs, HUVECs, and RAW264.7 cells was determined using a CCK-8 assay kit. BMSCs, HUVECs, and RAW264.7 cells were seeded onto TA, AE-MXene prepared in Examples 1, 5-9, and AE prepared in Comparative Example 2, respectively, and cultured in a 37°C, 5% CO2 incubator. Then, 1 / 10 of the culture medium volume of CCK-8 reagent was added to each well, and the cells were cultured for another 4 h. The absorbance was measured at 450 nm, and cell viability was calculated. Figure 2 As shown. By Figure 2 It was found that when the concentration of MXene dispersion was 0.025–0.2 mg / mL, the viability of the three cell types was the highest, and none of them showed significant toxicity. However, cell viability decreased when the concentration of MXene dispersion exceeded 0.2 mg / mL. Therefore, the concentration of MXene dispersion should be controlled below 0.2 mg / mL.

[0036] Figure 3 Optical images of AE-MXene prepared for TA and Examples 1, 5-7, and AE prepared for Comparative Example 2. From Figure 3 As can be seen, both TA and AE surfaces are white, while titanium sheets loaded with different concentrations of MXene all exhibit a characteristic blue-purple luster, indicating that MXene nanosheets have been successfully attached to the surface of the titanium sheets.

[0037] Figure 4 The staining results of BMSCs, HUVECs, and RAW264.7 cells with TA, AE-MXene prepared in Examples 1 and 7, and AE prepared in Comparative Example 2. BMSCs, HUVECs, and RAW264.7 cells were seeded onto TA, AE-MXene prepared in Examples 1 and 7, and AE prepared in Comparative Example 2, respectively, and cultured in a 37°C, 5% CO2 incubator for [number] days. Live / dead cell staining was then performed using the Calcein-AM / PI cell viability kit. After incubation for 30 min, cell images were captured using a fluorescence microscope. Cells stained with Calcein-AM appeared green, and cells stained with PI appeared red. Live / dead cells were counted, and statistical graphs were plotted. Figure 4 It can be seen that when the concentration of MXene dispersion is 0.2 mg / mL, cell apoptosis increases.

[0038] Zeta potential tests were performed on the MXene dispersions prepared in Examples 1, 5-7, as follows: Figure 5 As shown. By Figure 5 It is known that MXene nanosheets have inherent electronegativity, and the amplitude of the negative surface potential increases synchronously with the increase of concentration.

[0039] DLS particle size analysis was performed on the MXene dispersions prepared in Examples 1, 5-7, as follows: Figure 6 As shown. By Figure 6 It is evident that the average particle size of MXene nanosheets decreases with increasing concentration. The stability of nanoparticles is inversely proportional to their particle size. Larger particles tend to have lower surface charge and are more prone to aggregation. Increasing MXene concentration leads to a decrease in particle size, an increase in specific surface area, and an increase in surface charge; these factors collectively promote a more stable dispersion system.

[0040] The above Zeta potential and DLS particle size test results confirm that MXene nanosheets have excellent surface charge properties and good colloidal stability.

[0041] Figure 7 SEM images of TA, AE-MXene prepared in Example 1, TA-MXene prepared in Comparative Example 1, and AE prepared in Comparative Example 2. From... Figure 7 It can be seen that when MXene is loaded onto the TA surface without alkali etching, its distribution is uneven; while the AE surface after alkali etching can promote more uniform and stable deposition of MXene, forming a continuous coating that enhances the adhesion of MXene.

[0042] Figure 8 XRD patterns of TA-MXene prepared for TA and Comparative Example 1. Figure 8 It can be seen that the MXene coating exhibits a significant diffraction peak at approximately 6.8°, indicating that it has the (002) crystal plane characteristic of MXene, which typically appears in the 6-10° range.

[0043] Figure 9 XPS plots of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2. From Figure 9 It can be seen that a significant F 1s peak was observed near 683.7 eV for AE-MXene, indicating the presence of fluorine-containing functional groups on the MXene surface, which is related to its strong electronegativity, further confirming the successful loading of MXene.

[0044] Figure 10 The AFM image of AE-MXene prepared in Example 1. Figure 10 It can be seen that MXene is uniformly distributed on the AE surface, with an average height distribution of approximately 5–6 nm. The rough surface is more conducive to osteoblast differentiation and bone integration.

[0045] Figure 11 Surface roughness statistics of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2. Figure 11It can be seen that the arithmetic mean roughness (Sa) and root mean square roughness (Sq) of AE are higher than those of TA, while the Sa and Sq of AE-MXene are higher than those of AE. This indicates that alkaline etching can improve the surface roughness of titanium sheets, and composite MXene on the surface of alkaline-etched titanium sheets can further improve the surface roughness.

[0046] Increased surface roughness is generally associated with enhanced surface hydrophilicity. Surfaces with smaller contact angles are considered hydrophilic, while larger contact angles indicate hydrophobicity. Figure 12 The water contact angle test results for TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2. Figure 12 It is evident that AE has a smaller water contact angle compared to TA, indicating that alkaline etching can improve the hydrophilicity of titanium sheets; while AE-MXene has an even smaller water contact angle, indicating that MXene composite on the alkaline-etched titanium sheet surface can further improve hydrophilicity. Given that enhanced hydrophilicity has been proven to promote osteogenic differentiation and early osseointegration, the AE-MXene surface prepared in this invention may have even better osseointegration potential.

[0047] In vitro evaluation of the anti-inflammatory, osteogenic, and angiogenic effects of AE-MXene: Macrophage polarization and function can prevent peri-implant inflammatory osteolysis. To assess the immunomodulatory effects of MXene, this invention examined the influence of MXene on the inflammatory phenotype in cultured RAW264.7 cells.

[0048] RAW264.7 cells were seeded in 6-well plates containing TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2, and cultured to 80% confluence. Cells were then stimulated with lipopolysaccharide (LPS, 100 ng / mL) for 24 h. After culture, cells were collected, total RNA was extracted, and reverse transcription was performed in the cells using the PrimeScript RT kit. Reverse transcriptase-polymerase chain reaction (RT-qPCR) was performed using a real-time quantitative PCR system (Light Cycler 480II, Roche) and SYBR Premix Ex Taq II. The reaction mixture consisted of 5 μL SYBR mixture, 3 μL double-distilled water, 1 μL primers, and 1 μL cDNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was an internal reference gene, and relative gene expression levels were calculated using 2⁻¹. ΔΔCt The method was determined. Primer sequences are shown in Table 1. Each group had three replicates. Figure 13 The effects of TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2 on the mRNA expression of pro-inflammatory cytokines. From Figure 13It can be seen that, compared with TA, AE-MXene (0.1 mg / mL) significantly inhibited the mRNA expression of pro-inflammatory cytokines (including Il1b, Il6, Il23a and Il11), while AE-MXene (0.2 mg / mL) significantly inhibited the mRNA expression of Il6 and Il23a, indicating that AE-MXene can inhibit the expression of macrophage inflammatory factors.

[0049] Table 1 Primers used in real-time quantitative PCR Primer sequence Forward: 5ʹ-AGGTCGGTGTGAACGGATTTG-3ʹ Reverse: 5ʹ-TGTAGACATGTAGTTGAGGTCA-3ʹ Forward: 5ʹ-GGAGCGAGATCCCTCCAAAAT-3ʹ Reverse: 5ʹ-GGCTGTTGTCATACTTCTCATGG-3ʹ Forward: 5ʹ-TGAGCACCTTCTTTTCCTTCATC-3ʹ Reverse: 5ʹ-TGTCTAATGGGAACGTCACACAC-3ʹ Forward: 5ʹ-GAGCCCACCAAGAACGATAG-3ʹ Reverse: 5ʹ-TTTCCACGATTTCCCAGAGA-3ʹ Forward: 5ʹ-TGTTCTCCTAACCCGATCCCT-3ʹ Reverse: 5ʹ-CAGGAAGCTGCAAAGATCCCA-3ʹ Forward: 5ʹ-GACTCTTGCGTCAACTTCAAGG-3ʹReverse: 5ʹ-CAGGCTGTCTTTTGTCAACGA-3ʹ Forward: 5ʹ-ATGCTGGATTGCAGAGCAGTA-3ʹReverse: 5ʹ-ACGGGGCACATTAATTTTTAGTCT-3ʹ Forward: 5ʹ-CTCTGTTCAGCTATTGGACGC-3ʹReverse: 5ʹ-CGGAATTTCTGGGATTCAGCTTC-3ʹ Forward: 5ʹ-CTCCAAGCCAAAGTCCTTAGAG-3ʹ Reverse: 5ʹ-AGGAGCTGTCATTAGGGACATC-3ʹ Forward: 5ʹ-TCGCTGATGCACTGCCTATG-3ʹ Reverse: 5ʹ-GAGAGGTCCACAGAGCTGATT-3ʹ Forward: 5ʹ-CCAATCCAGCTAACTATCCCTCC-3ʹ Reverse: 5ʹ-ACCCAGTAGCAGTCATCCCA-3ʹ Forward: 5ʹ-ATGCTTCATTCGCCTCACAAA-3ʹ Reverse: 5ʹ-GCACTCACTGACTCGGTTGG-3ʹ Forward: 5ʹ-GCAATAAGGTAGTGAACAGACTCC-3ʹ Reverse: 5ʹ-CCATAGATGCGTTTGTAGGCGG-3ʹ Forward: 5ʹ-GCTCCTCTTAGGGGCCACT-3ʹ Reverse: 5ʹ-CCACGTCTCACCATTGGGG-3ʹ Forward: 5ʹ-ATGGCGTCCTTCTCTGCTTG-3ʹ Reverse: 5ʹ-TGAAAGGTCAGCGTATGGCTT-3ʹ Forward: 5ʹ-CCAACTCTTTTTGTGCCAGAGA-3ʹ Reverse: 5ʹ-GGCTACATTGGTGTTGAGCTTTT-3ʹ Forward: 5ʹ-AGGGCAGAATCATCACGAAGT-3ʹ Reverse: 5ʹ-AGGGTCTCGATTGGATGGCA-3ʹ Forward: 5ʹ-CTGGGCGTTTTGTTGTTGGTC-3ʹ Reverse: 5ʹ-GGTTTGGCATCATAGTGCTGG-3ʹ RAW264.7 cells were seeded in 6-well plates containing TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2, respectively, and cultured to 80% confluence. Cells were then stimulated with LPS (100 ng / mL) for 24 h. After culture, cells were collected and antibodies against F4 / 80, anti-CD11B, anti-CD86 (M1 type), and anti-CD206 (M2 type) were added. Marker expression levels were detected using a FACScan flow cytometer (BDBiosciences, CA), and cell polarization was calculated using FlowJo software. Each sample group underwent three independent experiments. Figure 14 To analyze the effects of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2 on the expression of M1 marker (CD86) and M2 marker (CD206) genes in RAW264.7 cells under inflammatory conditions using flow cytometry. Figure 14 It can be seen that the proportion of CD86-positive cells and the mean fluorescence intensity (MFI) of CD86 were not significantly different among the LPS group, TA group, AE group, and AE-MXene group; however, the proportion of CD206-positive cells increased from 4.79±0.65% in the LPS group to 13.23±1.11% in the AE-MXene group, and the MFI value of CD206 increased from 56.33±4.31 in the LPS group to 82.8±5.9 in the AE-MXene group. This indicates that AE-MXene can promote M2 polarization of macrophages, inhibit inflammation, and construct an immune microenvironment conducive to regeneration.

[0050] Figure 15 To analyze the effects of TA, AE-MXene prepared in Example 1, and AE prepared in Comparative Example 2 on the expression of M1 markers (Il11, Il18, Il23a) and M2 markers (Mrc1, Arg1, Pparg, Retnla) in RAW264.7 cells under inflammatory conditions using RT-qPCR. Figure 15It can be seen that, compared with TA and AE, AE-MXene significantly promoted the downregulation of M1 markers and the upregulation of M2 markers. This result further confirms that AE-MXene can promote M2 polarization of macrophages, thereby reducing inflammation and supporting bone regeneration.

[0051] BMSCs were seeded onto TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2, respectively, and then cultured in conventional culture medium and osteogenic induction medium (Oricell, China). On day 7, cells were lysed with RIPA lysis buffer (Bristol-Myers Squibb, China) (without inhibitors), and the supernatant was collected to determine ALP activity. The ALP detection kit was followed according to the instructions: chromogenic substrate solution was added to the sample, incubated at 37°C for 30 min, and absorbance was measured at 405 nm. The total protein content of each group was detected using the BCA protein assay kit (Bio-Thera Solutions, China), and ALP levels were normalized. Each sample was tested three times independently. Figure 16 The effects of TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2 on ALP activity in BMSCs cells were investigated. NM served as a negative control using standard culture medium, and OCM served as a positive control using osteogenic induction culture medium. Figure 16 It can be seen that AE-MXene (0.1 mg / mL) significantly enhances osteogenic potential.

[0052] Figure 17 To analyze the effects of TA, AE-MXene prepared in Examples 1, 5-7, and AE prepared in Comparative Example 2 on the expression of osteogenic genes (Runx2, Bglap, Col1a1, Sp7, and Alpl) in BMSCs cells by RT-qPCR. Figure 17 It was observed that on day 7, the expression of the Sp7 gene was upregulated by AE-MXene (0.1 mg / mL), while the expression of the Bglap and Col1a1 genes was downregulated by AE-MXene (0.2 mg / mL); on day 14, the mRNA levels of the Runx2, Bglap, Col1a1, and Alpl genes were significantly increased by AE-MXene (0.05 mg / mL) and AE-MXene (0.1 mg / mL). These findings suggest that AE-MXene (0.1 mg / mL) has enhanced osteogenic activity.

[0053] Bone formation around implants is closely related to angiogenesis, which provides essential nutrients and oxygen for osteogenic processes and supports the proliferation and differentiation of related cell types. This invention uses human umbilical vein endothelial cells (HUVECs) to conduct lumen formation experiments to evaluate the pro-angiogenic potential of AE-MXene.

[0054] Sterile TA, AE-MXene prepared in Examples 1, 6, and 7, and AE prepared in Comparative Example 2 were added to 24-well plates, respectively. Then, 250 μL of L Atrigel coating solution was added to each well, and gelation was carried out at 37°C for 30 min. HUVECs cells were starved for 8 h and then cultured at 1.5 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 1000 μg / mL on Matrigel substrate and cultured for 6 h. Calcein-AM dye was then added to a final concentration of 2 μg / mL, and the cells were incubated at 37°C in the dark for 30 min. Images of the tubular structures were captured using an inverted fluorescence microscope (Leica, Germany), and parameters such as the number of branch nodes and tube length were analyzed using ImageJ software. Each sample group underwent three independent experiments. Figure 18 Immunofluorescence images of HUVECs cells treated with TA and AE-MXene prepared in Examples 1, 6, and 7, respectively, and AE prepared in Comparative Example 2. Figure 19 To quantitatively analyze the network structure and branch number in immunofluorescence images using ImageJ software. Figure 20 To analyze the expression of VEGF and ANG in HUVEC cells using RT-qPCR. Figure 19 It was found that, compared with TA, AE-MXene (0.1 mg / mL) significantly promoted lumen formation; and with increasing MXene concentration, the formed network structure and branches significantly increased, reaching a peak at 0.1 mg / mL. Furthermore, from Figure 20 It was found that VEGF gene expression was significantly upregulated in the presence of MXene, with AE-MXene (0.1 mg / mL) showing the largest increase. Although alkaline etching of the titanium sheet inhibited ANG expression, AE-MXene (0.1 mg / mL) reversed this inhibitory effect. In conclusion, AE-MXene (0.1 mg / mL) promoted angiogenesis.

[0055] Improvements in technologies such as liquid chromatography-tandem mass spectrometry (LC-MS / MS) have enhanced the sensitivity, accuracy, and throughput of proteomics studies, providing crucial insights into cell function, signal transduction pathways, and disease mechanisms. This invention employs a data-independent acquisition (DIA)-based quantitative proteomics approach to identify molecular pathways regulated by AE-MXene. RAW264.7 cells were seeded in 6-well plates containing TA and AE-MXene prepared in Example 1, respectively, and cultured to 80% confluence. Cells were then stimulated with LPS (100 ng / mL) for 24 h. After culture, cells were harvested and processed according to… Figure 21 Mass sequencing was performed, and each sample group underwent three independent experiments. Functional enrichment analysis revealed autophagy as a key pathway. Figure 22This finding is consistent with the key role autophagy plays in maintaining macrophage homeostasis and mediating stress responses.

[0056] Representative images of autophagic flux visualization using the mRFP-GFP-LC3 combined fluorescent reporter system. RAW264.7 cells stably expressing this reporter gene were seeded onto TA and AE-MXene surfaces prepared in Example 1, respectively. According to each group's requirements, cells were pretreated with chloroquine (CQ, 5 μM) or 3-methyladenine (3-MA, 5 mM) for 2 h, and then stimulated with fresh medium containing LPS (100 ng / mL) for 24 h. Autophagosomes (APs) appeared as small yellow dots (mRFP... + GFP + Autolysosomes (ALs) only show small red fluorescent dots (mRFPs). + GFP – ). Figure 23 Representative fluorescence images and corresponding quantitative statistical analysis graphs of AP number, AL number, and AL / AP ratio are provided. Each sample group underwent three independent experiments. Figure 23 It can be seen that AE-MXene increased the number of yellow (autophagosomes) and red (autolysosomes) spots, while CQ blocked the degradation of autophagosomes (leading to the accumulation of yellow spots), and 3-MA inhibited the formation of autophagosomes and autolysosomes. This indicates that AE-MXene promoted the activation of the complete autophagic flux of macrophages.

[0057] To verify the role of autophagy in the biological effects of AE-MXene, this invention uses macrophage culture medium to simulate paracrine interactions between macrophages and target cells. Figure 24 RAW264.7 cells and THP-1 cells were seeded in 6-well plates containing TA and AE-MXene prepared in Example 1, respectively, and cultured to 80% confluence. Cells were pretreated with CQ (5 μM) for 2 h, then stimulated with fresh medium containing LPS (100 ng / mL) for 24 h. The collected supernatant was used as the corresponding macrophage conditioned medium (CM).

[0058] BMSCs were seeded into 24-well plates. After cell adhesion, the culture medium (CM) was replaced, and the cells were cultured for 7 days, with fresh CM replaced every 2-3 days. On day 7, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, followed by ALP staining and Alizarin Red S staining. The procedure was performed according to the kit instructions: incubation at room temperature for 30 min. After the reaction was terminated, cells were washed with PBS. Each sample was tested three times independently. Figure 25ALP and Alizarin Red S staining results demonstrated that AE-MXene significantly enhanced the osteogenic potential of BMSCs, with a more pronounced effect compared to TA. Notably, this osteogenic-promoting effect was reversed after pretreatment of cells with the autophagy inhibitor CQ.

[0059] BMSCs were seeded in 24-well plates. After cell adhesion, the culture medium (CM) was replaced, and the cells were cultured for 7 days, with fresh CM replaced every 2-3 days. On day 7, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Tween X-100 for 10 min, and mounted for 1 h. Primary antibodies against actin, alkaline phosphatase, and osteocalcin were added, and the cells were incubated overnight. The next day, the corresponding fluorescently labeled secondary antibodies (Alexa Fluor 488 or 594) were added, and the cells were incubated in the dark for 1 h. After staining the cell nuclei with DAPI, images were acquired using a confocal laser scanning microscope, and the fluorescence intensity was quantitatively analyzed using ImageJ software. Each sample was subjected to three independent experiments. Immunofluorescence analysis further confirmed that AE-MXene upregulated the expression of early (ALP) and intermediate (osteocalcin, OCN) osteogenic markers in BMSCs. CQ pretreatment significantly attenuated the upregulation of osteogenic markers induced by AE-MXene. Figure 26 In summary, AE-MXene promotes osteogenic differentiation under inflammatory conditions, and this effect is achieved by promoting autophagy in macrophages.

[0060] use Figures 18-20 Corresponding lumen formation assays and RT-qPCR assays were used to assess the effect of macrophage autophagy on the pro-angiogenic potential of AE-MXene. The difference was that fresh CM was replaced every 2-3 days during the culture period. Figure 27 Immunofluorescence images of HUVECs cells after CM treatment and quantitative analysis of the network structure and branch number in the immunofluorescence images. Figure 28 To analyze the expression of VEGF and ANG in HUVECs cells treated with CM using RT-qPCR. Figure 27 As can be seen, compared with TA, AE-MXene significantly increased the number of network structures and segments, indicating enhanced vascular network formation. Pretreatment with CQ effectively reversed the pro-angiogenic effect of AE-MXene. RT-qPCR analysis further showed that AE-MXene upregulated the expression of angiogenesis-related genes, including ANG and VEGF, while CQ pretreatment inhibited this upregulation. Figure 28 In summary, AE-MXene promotes angiogenesis and differentiation under inflammatory conditions, and this effect is achieved by promoting autophagy in macrophages.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a titanium sheet / MXene composite material, characterized in that, Includes the following steps: (1) Acid etching, centrifugation, washing, ultrasonic peeling and drying of titanium aluminum carbide powder were performed to obtain MXene nanosheets; (2) MXene nanosheets were dispersed in deionized water to obtain an MXene dispersion; (3) The MXene dispersion was coated onto the surface of the alkali-etched titanium sheet and dried to obtain the titanium sheet / MXene composite material.

2. The preparation method according to claim 1, characterized in that: The acid etching uses a LiF / HCl mixed solution as the etching solution, wherein the molar ratio of LiF to HCl is 1:(2~3).

3. The preparation method according to claim 1, characterized in that: The ratio of the titanium aluminum carbide powder to the LiF / HCl mixed solution is 1 g : (10~20) mL.

4. The preparation method according to claim 1, characterized in that: The acid etching temperature is 30~40℃ and the time is 24~28h.

5. The preparation method according to claim 1, characterized in that: The ultrasonic ablation is performed at a frequency of 20-40 kHz, a power of 100-500 W, and a duration of 0.5-2 h.

6. The preparation method according to claim 1, characterized in that: The concentration of the MXene dispersion is 0.025~10 mg / mL.

7. The preparation method according to claim 1, characterized in that: The alkaline etching uses potassium hydroxide solution as the etching solution with a concentration of 1~5 M; Preferably, the alkaline etching temperature is 30~50℃ and the time is 1~8h.

8. The preparation method according to claim 1, characterized in that: The coating method is at least one of drip coating, roller coating, brush coating, spray coating, and dip coating.

9. A titanium sheet / MXene composite material prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the titanium sheet / MXene composite material of claim 9 as a medical implant.

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