Mxene / Zn nano-coating modified CF / PEEK as well as preparation method and application thereof

Abstract

The invention discloses Mxene / Zn nano coating modified CF / PEEK as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, pretreating a CF / PEEK stent; s2, sulfonating the surface of the CF / PEEK stent; s3, a PEI-TA adhesive layer is constructed; s4, a CF / PEEK-MXene material is prepared; and S5, preparing the CF / PEEK-MXene coated Zn material. Through the synergistic effect of promoting osteogenesis and angiogenesis, the MXene / Zn nano-coating modified CF / PEEK stent can effectively solve the problem of mismatch of multiple biological interfaces of mechanical support-nutrition transport-cell recruitment in large-section bearing bone defect repair. The gradient mechanical adaptation characteristic of the stent can provide stable mechanical support, meanwhile, good mechanical matching with surrounding bone tissues is achieved, and the stress shielding effect is avoided. And the blood vessel-osteogenesis coupling activity ensures that the collection of cells, the transportation of nutrition and the formation of new bones can be orderly carried out in the bone repair process, so that the success rate of large-section bearing bone defect repair is greatly improved.

Description

Technical Field

[0001] The present invention relates to MXene / Zn nano-coating-modified CF / PEEK and a preparation method and application thereof, which are particularly suitable for repairing large-segment load-bearing bone defects and belong to the technical field of biomedical engineering. Background Art

[0002] Large, weight-bearing bone defects are a major challenge in orthopedic clinical treatment, commonly occurring in situations such as severe trauma, tumor resection, and congenital bone dysplasia. These defects not only affect the patient's limb function but also reduce their quality of life and even threaten their health. Currently, these defects are primarily repaired using autologous bone transplantation, allogeneic bone transplantation, and synthetic bone substitute materials.

[0003] Autologous bone transplantation is considered the "gold standard" for bone defect repair due to its advantages such as good osteoconductivity, osteoinductivity, and lack of immune rejection. However, this method is limited in the bone donor area, causes additional trauma and pain to the patient during bone harvesting, and may cause complications such as infection, making it difficult to meet the needs of repairing large, load-bearing bone defects. Although allogeneic bone transplantation can address the issue of insufficient bone supply to a certain extent, it is subject to immune rejection and the risk of disease transmission. It also requires long-term use of immunosuppressants after surgery, increasing treatment costs and potential health risks for patients.

[0004] Synthetic bone replacement materials, such as metal implants, ceramic materials, and polymer materials, offer advantages such as a wide range of sources and customizability. Among them, CF / PEEK (carbon fiber reinforced polyetheretherketone) composites, with their excellent mechanical properties, biocompatibility, and low elastic modulus, are more compatible with human bone tissue and exhibit considerable potential for application in bone repair. However, single CF / PEEK materials suffer from insufficient surface bioactivity and weak binding to bone tissue, making them ineffective in promoting the adhesion, proliferation, and differentiation of bone cells and achieving good bone integration. Furthermore, during the repair of large, load-bearing bone defects, they face challenges with multiple biointerface mismatches, such as mechanical support, nutrient transport, and cell recruitment, making it difficult to simultaneously meet the mechanical and biological performance requirements of bone repair.

[0005] Therefore, developing a method that can improve the surface properties of CF / PEEK materials and construct a multi-level bionic scaffold system with good mechanical adaptation properties and bioactivity to overcome the problem of multiple biological interface mismatch in the repair of large load-bearing bone defects has become a key issue that needs to be urgently addressed in the current field of bone tissue engineering.

[0006] In response to the above problems, this application is filed. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention takes CF / PEEK as the research object, aiming to improve the surface properties of CF / PEEK material by constructing a specific nano-coating on its surface, and construct a multi-level bionic scaffold system with gradient mechanical adaptation properties and vascular-osteogenic coupling activity, thereby breaking through the multiple biological interface mismatch problem of mechanical support-nutrient transport-cell recruitment in the repair of large load-bearing bone defects.

[0008] The first object of the present invention is to provide a method for preparing CF / PEEK modified with a MXene / Zn nanocoating, comprising the following steps: S1: CF / PEEK scaffold pretreatment The CF / PEEK scaffold was ultrasonically cleaned with deionized water for 10 minutes and then dried to remove impurities on the CF / PEEK scaffold surface, providing a clean surface for subsequent treatment and facilitating better bonding between the subsequent coating and the substrate. S2: CF / PEEK scaffold surface sulfonation The CF / PEEK scaffold dried in step S1 was added to concentrated sulfuric acid with a mass concentration of 90%. Continuous magnetic stirring was applied to ensure that the CF / PEEK scaffold was suspended in the sulfuric acid solution. After full and uniform contact for 3 minutes, the scaffold was removed and immediately placed in deionized water for ultrasonic cleaning for 3 minutes, and then dried. Sulfonic acid groups were introduced on the surface of the CF / PEEK through sulfonation treatment to increase surface activity and improve the bonding strength between the subsequent coating and the substrate, thus solving the problem of weak bonding between the single CF / PEEK material and the coating.

[0009] S3: Construction of PEI-TA Adhesion Layer Polyethyleneimine (PEI) and tannic acid (TA) were dissolved in deionized water to a concentration of 40 mg / mL for both PEI and TA. The dried material from step S2 was then added to 15 mL of PEI solution, ensuring that the solution completely submerged the material and thoroughly stirred. After stirring for 10 minutes, 15 mL of TA solution was slowly added. After the addition was complete, the mixture was allowed to react for 30 minutes. When no obvious flocs were present, the mixed solution was titrated with 0.1 mol / L HCl solution to a pH of 4.2 ± 0.5. The mixture was sealed and stored overnight. The PEI-TA adhesion layer provided a good foundation for the subsequent adhesion of the MXene coating, further enhancing the stability of the bonding between the coating and the substrate.

[0010] S4: Preparation of CF / PEEK-MXene Materials The material obtained in step S3 was removed and rinsed with deionized water and dried. Then, 40 mL of a 0.25 mg / mL MXene solution was added and placed in a 37°C constant temperature shaker for 24 hours to obtain a CF / PEEK-MXene material and a MXene solution system. MXene has excellent conductivity, biocompatibility, and bioactivity, and can promote cell adhesion, proliferation, and differentiation, thereby improving the problem of insufficient surface bioactivity of the CF / PEEK material.

[0011] S5: Preparation of CF / PEEK-MXene@Zn materials To the MXene solution from step S4, 40 mL of deionized water was added for dilution. 137.4 mg of zinc acetate was weighed and added to the mixed solution, stirring continuously for 1 hour. Then, 624 mg of thioacetamide was added and stirred for 10 minutes. After sufficient reaction, the mixed solution and the materials were transferred to a hydrothermal autoclave and heated at 60°C for 4 hours. After cooling, the material was removed to obtain CF / PEEK modified with a MXene / Zn nanocoating, i.e., CF / PEEK-MXene@Zn material. Zn, which promotes bone growth and has antibacterial properties, synergizes with MXene to further enhance the material's bioactivity and bone repair ability, strengthening its overall performance for repairing large, load-bearing bone defects.

[0012] Preferably, the drying conditions in steps S1, S2, and S4 are: temperature 60-80°C, time 2-4h.

[0013] Using the above technical solution, the present invention constructs a MXene / Zn nanocoating on the surface of the stent through a low-temperature hydrothermal method, which has multiple functional effects: (1) Osteogenesis: MXene / Zn nano-active coating has a temporal regulatory effect on the proliferation, adhesion morphology and osteogenic differentiation of rBMSC cells (rat bone marrow mesenchymal stem cells). In the early stage of cell proliferation, the bioactive signals released by MXene and Zn elements in the coating can stimulate rBMSC cells to quickly enter the proliferation stage and increase the number of cells. Over time, the coating will further guide the adhesion morphology of the cells, allowing them to better fit on the surface of the scaffold, laying the foundation for subsequent osteogenic differentiation. In the osteogenic differentiation stage, MXene and Zn elements work synergistically to upregulate the expression of osteogenesis-related genes and promote the secretion of bone matrix proteins such as osteocalcin and osteopontin, thereby accelerating the formation of new bone.

[0014] (2) Promoting angiogenesis: MXene / Zn nanocoating can significantly promote the proliferation, migration and formation of vascular endothelial cells and vascular-like structures. The good electrical conductivity of MXene can simulate the electrical microenvironment of the extracellular matrix, activate the signal transduction pathways in vascular endothelial cells, and promote cell proliferation and migration. At the same time, the Zn element can regulate the expression of angiogenesis-related factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), etc. These factors can further attract vascular endothelial cells to gather around the stent and induce them to form vascular-like structures. In addition, the porous structure of the nanocoating provides physical support and channels for the growth of blood vessels, which is conducive to the growth and distribution of new blood vessels inside and around the stent, thereby providing sufficient nutrition and oxygen supply for bone tissue repair and accelerating the repair process of bone defects.

[0015] The second object of the present invention is to provide CF / PEEK modified with MXene / Zn nano-coating, wherein the CF / PEEK-MXene@Zn material forms a specific MXene / Zn nano-coating structure on the surface of CF / PEEK through the above preparation steps.

[0016] Preferably, a layer-by-layer progressive MXene / Zn nanodot active coating is formed on the surface of the CF / PEEK-MXene@Zn material, which has a multi-level bionic structure with gradient mechanical adaptation properties and angiogenesis-osteogenesis coupling activity.

[0017] The third object of the present invention is to provide an application of CF / PEEK-MXene@Zn material in the repair of large load-bearing bone defects.

[0018] By employing the aforementioned technical solution, and through the synergistic effects of osteogenesis and angiogenesis, the MXene / Zn nanocoated CF / PEEK scaffold of the present invention can effectively address the multiple bio-interface mismatches of mechanical support, nutrient transport, and cell recruitment in the repair of large, load-bearing bone defects. The scaffold's gradient mechanical adaptation properties provide stable mechanical support while achieving good mechanical matching with the surrounding bone tissue, avoiding stress shielding effects. The angiogenic-osteoblastic coupling activity ensures that cell recruitment, nutrient transport, and new bone formation proceed in an orderly manner during bone repair, significantly improving the success rate of repairing large, load-bearing bone defects.

[0019] Beneficial effects of the present invention: (1) The present invention proposes a method for preparing MXene / Zn nano-coating modified CF / PEEK, which constructs a multi-level bionic scaffold system by introducing a layer-by-layer progressive MXene / Zn nano-dot active coating on the surface of the CF / PEEK scaffold.

[0020] (2) Through the synergistic effects of promoting osteogenesis and angiogenesis, the MXene / Zn nanocoating modified CF / PEEK scaffold of the present invention can effectively solve the multiple biological interface mismatch problems of mechanical support, nutrient transport and cell recruitment in the repair of large load-bearing bone defects. The gradient mechanical adaptation characteristics of the scaffold can achieve good mechanical matching with the surrounding bone tissue while providing stable mechanical support, avoiding the stress shielding effect. The angiogenesis-osteogenesis coupling activity ensures that cell recruitment, nutrient transport and new bone formation can proceed in an orderly manner during the bone repair process, greatly improving the success rate of repairing large load-bearing bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The appearance, morphology and characteristics of composite bionic scaffold samples in different groups are different.

[0022] Figure 2 SEM was used to present the microscopic morphology of each group of composite bionic scaffolds.

[0023] Figure 3 This is the particle size distribution diagram of ZnS nanodots.

[0024] Figure 4 The XPS full spectrum data and element chemical state information of each group of composite bionic scaffolds.

[0025] Figure 5 CCK8 cell proliferation data of BMSCs under treatment of different groups of composite biomimetic scaffolds.

[0026] Figure 6 The results of quantitative analysis of ALP staining of BMSCs treated with composite bionic scaffolds in different groups.

[0027] Figure 7 RUNX2 gene expression data in BMSCs treated with different groups of composite bionic scaffolds.

[0028] Figure 8 The OCN gene expression data in BMSCs treated with different groups of composite bionic scaffolds.

[0029] Figure 9 ALP gene expression data in BMSCs treated with different groups of composite bionic scaffolds.

[0030] Figure 10 CCK8 cell proliferation data of HUVECs treated with different groups of composite biomimetic scaffolds.

[0031] Figure 11 The expression of HIF-1α gene in HUVECs treated with composite bionic scaffolds in different groups.

[0032] Figure 12The expression of VEGF gene in HUVECs treated with composite bionic scaffolds in different groups.

[0033] Figure 13 The expression of CD31 gene in HUVECs under treatment of different groups of composite bionic scaffolds. DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0035] Example 1 The preparation method of MXene / Zn nano-coating modified CF / PEEK comprises the following steps: S1: CF / PEEK scaffold pretreatment The CF / PEEK scaffold was ultrasonically cleaned with deionized water for 10 min and then dried; S2: CF / PEEK scaffold surface sulfonation The CF / PEEK scaffold dried in step S1 was added to 90% concentrated sulfuric acid. Magnetic stirring was continued to ensure that the CF / PEEK scaffold was suspended in the sulfuric acid solution. After sufficient and uniform contact for 3 minutes, the scaffold was removed and immediately ultrasonically cleaned in deionized water for 3 minutes, and then dried. S3: Construction of PEI-TA Adhesion Layer Dissolve polyethyleneimine (PEI) and tannic acid (TA) in deionized water to a concentration of 40 mg / mL each. Then, add the material dried in step S2 to 15 mL of PEI solution, ensuring that the solution completely submerges the material and stir thoroughly. After stirring for 10 minutes, slowly add 15 mL of TA solution. After the addition is complete, allow the mixture to react for 30 minutes. When no flocs are evident, adjust the pH of the mixed solution and seal overnight. S4: Preparation of CF / PEEK-MXene Materials The material obtained in step S3 was taken out and rinsed with deionized water, dried, and then 40 mL of 0.25 mg / mL MXene solution was added. The mixture was placed in a 37°C constant temperature shaker for 24 h to obtain a CF / PEEK-MXene material and a MXene solution system. S5: Preparation of CF / PEEK-MXene@Zn materials Add 40 mL of deionized water to the MXene solution system in step S4 to dilute it, weigh 137.4 mg of zinc acetate and add it to the mixed solution and continue stirring for 1 hour, then add 624 mg of thioacetamide and stir for 10 minutes. After sufficient reaction, transfer the mixed solution and the material to a hydrothermal autoclave and heat at 60 ° C for 4 hours. After cooling, take out the material to obtain CF / PEEK modified with MXene / Zn nanocoating, that is, CF / PEEK-MXene@Zn material.

[0036] In this embodiment, the drying conditions in step S1 are: temperature 60° C., time 2 h.

[0037] In this embodiment, the drying conditions in step S2 are: 80° C., time 2 h.

[0038] In this embodiment, the drying conditions in step S3 are: 80° C., time 4 hours.

[0039] In this embodiment, in step S3, 0.1 mol / L HCl solution is used to titrate the mixed solution to pH=4.2.

[0040] Example 2 The difference between this embodiment and embodiment 1 is that: In this embodiment, the drying conditions in step S1 are: temperature 70° C., time 3 hours.

[0041] In this embodiment, the drying conditions in step S2 are: 60° C., time 4 hours.

[0042] In this embodiment, the drying conditions in step S3 are: 70° C., time 3 hours.

[0043] In this embodiment, in step S3, 0.1 mol / L HCl solution is used to titrate the mixed solution to pH=4.5.

[0044] Other details are the same as in Example 1.

[0045] Example 3 The difference between this embodiment and embodiment 1 is that: In this embodiment, the drying conditions in step S2 are: 70° C., time 4 hours.

[0046] In this embodiment, in step S3, 0.1 mol / L HCl solution is used to titrate the mixed solution to pH=3.9.

[0047] Other details are the same as in Example 1.

[0048] Example 4 The difference between this embodiment and embodiment 1 is that: In this embodiment, the drying conditions in step S1 are: temperature 80° C., time 2 h.

[0049] In this embodiment, the drying conditions in step S2 are: 60° C., time 3 hours.

[0050] In this embodiment, the drying conditions in step S3 are: 70° C., time 2 h.

[0051] In this embodiment, in step S3, 0.1 mol / L HCl solution is used to titrate the mixed solution to pH=4.7.

[0052] Other details are the same as in Example 1.

[0053] Example 5 The difference between this embodiment and embodiment 1 is that: In this embodiment, the drying conditions in step S1 are: temperature 80° C., time 3 hours.

[0054] In this embodiment, in step S3, 0.1 mol / L HCl solution is used to titrate the mixed solution to pH=4.0.

[0055] Other details are the same as in Example 1.

[0056] Test Example 1 Test groups: Taking the CF / PEEK composite bionic scaffold as an example, the surface modified with Mxene nanocoating is recorded as CF / PEEK-Mxene, and the Zn nanocoating constructed on the surface of CF / PEEK-Mxene is recorded as CF / PEEK-Mxene@Zn.

[0057] Test method: Scanning electron microscopy (SEM) imaging revealed the micromorphology of each composite biomimetic scaffold. Three in vitro experimental scaffolds—CF / PEEK, CF / PEEK-MXene, and CF / PEEK-MXene@Zn—were subjected to a gold spraying treatment for approximately 30 seconds under vacuum conditions to enhance conductivity. The samples were then placed under SEM for surface micromorphology observation, with images of the material morphology captured at different magnifications.

[0058] Test results: refer to Figure 1 , showing the appearance of different groups of composite bionic scaffold samples and presenting the differences in external characteristics of each scaffold.

[0059] refer to Figure 2All groups of scaffolds showed a regular multi-level pore structure, and the surface topological characteristics and pore space distribution were highly consistent. The surface of the CF / PEEK group scaffold material was smooth, and a few carbon fiber shadows on the surface of the scaffold were evenly distributed inside the scaffold, and a few carbon fibers protruded from the surface of the scaffold; the MXene two-dimensional nanosheet coating on the surface of the CF / PEEK-MXene group composite scaffold was evenly distributed and the thickness was relatively uniform, with no obvious agglomeration.

[0060] refer to Figure 3 On the surface of the CF / PEEK-MXene@Zn composite scaffold, ZnS nanodots are distributed in the form of small spherical particles based on the MXene two-dimensional nanosheet coating. SEM measurements show that the diameter of the nanodots is approximately 129±25nm, and they grow evenly on the MXene surface without obvious agglomeration.

[0061] Test Example 2 Test group: Same as Test Example 1.

[0062] Test method: The chemical state of each element in the composite scaffold was analyzed by XPS. Three groups of in vitro experimental scaffold materials (CF / PEEK, CF / PEEK-MXene, and CF / PEEK-MXene@Zn) were placed in the sample chamber of the XPS analyzer to avoid the scattering of photoelectrons by gas molecules in the air. The vacuum degree of the sample chamber reached 2.0×10⁻. 7 After reaching mbar, the sample was transferred to the analysis chamber using a sample transfer procedure. Experimental parameters were set as follows: beam spot diameter 400 μm, operating voltage 12 kV, and filament current 6 mA. A monochromatized aluminum Kα (1486.6 eV) X-ray source was used to irradiate the sample, stimulating elemental emission of photoelectrons on the sample surface. An energy analyzer captured the photoelectrons emitted from the sample surface and measured their kinetic energy. The data were collected and exported in VGD format. Avantage software was used for charge correction and peak fitting, and Origin Pro 2008 software was used to plot the full XPS spectrum.

[0063] Test results: refer to Figure 4 Two main elements, C and O, were detected in the CF / PEEK scaffold; four elements, C, O, Ti, and F, were detected in the CF / PEEK-Mxene scaffold; and five elements, C, O, Ti, Zn, and S, were detected in the CF / PEEK-Mxene@Zn scaffold.

[0064] Test Example 3 Test group: Same as Test Example 1.

[0065] Test method: BMSCs treated with composite biomimetic scaffold materials in different groups were removed on day 1, 4, and 7, gently rinsed three times with PBS, and 1 mL of fresh culture medium and 100 μL of CCK-8 reagent were added to each well. The cells were incubated in the dark for 2 h (37°C) with tin foil. 200 μL of supernatant was transferred to a 96-well plate (Costar, 3599) and detected by dual wavelength (450 nm detection wavelength / 630 nm reference wavelength) using a microplate reader (BioTek Synergy H1). One-way analysis of variance (ANOVA) was performed using GraphPad Prism 8.0 and graphs were plotted.

[0066] Test results: refer to Figure 5 , CCK-8 results showed that on the first day, there was no significant difference in cell proliferation among the CF / PEEK, CF / PEEK-MXene, and CF / PEEK-MXene@Zn groups ( P> By day 4, the cell number in each group increased significantly. The OD values of the CF / PEEK-MXene and CF / PEEK-MXene@Zn groups were significantly higher than those in the CF / PEEK group (P < 0.05). There was no significant difference in OD values between the two groups. On day 7, the OD values of the CF / PEEK-MXene and CF / PEEK-MXene@Zn groups were significantly higher than those in the CF / PEEK group (P < 0.001).

[0067] Test Example 4 Test group: Same as Test Example 1.

[0068] Test method: This study quantitatively monitored the dynamic changes in enzyme activity during osteoblast differentiation using ALP. Following the kit instructions, the standard was serially diluted to obtain standard solutions of varying concentrations. Typically, 5-6 standard concentrations were set, such as 0, 5, 10, 20, 40, and 80 U / L. The standards of varying concentrations were added to the wells of the ELISA plate, with replicates set up for each concentration (usually 2-3 wells). Sample lysis: Add an appropriate amount of cell lysis buffer to the sample, homogenize appropriately, and centrifuge to obtain the supernatant for alkaline phosphatase detection. Sample loading: Add 50 μL of the prepared cell lysate or other sample to each well of the ELISA plate, with replicates set up for each sample. Ensure accurate loading to avoid errors.

[0069] Adding the chromogenic substrate: Add 50 μL of chromogenic substrate to each well of the ELISA plate to initiate the reaction. Mix thoroughly by gently pipetting with a pipette or using a shaker. Incubating: Place the plate in a 37°C (or the temperature specified by the kit) incubator for a specified period of time to allow the substrate to react with ALP to produce a colored product. The reaction time is typically between 5 and 10 minutes. If the alkaline phosphatase activity in the sample is low, the incubation time can be extended to 30 minutes. During the incubation process, avoid shaking the plate to prevent uneven reaction. Stopping the reaction: After the reaction time has expired, add the stop solution as directed in the kit instructions to terminate the reaction. Add the stop solution quickly and accurately to ensure that the reaction in all wells stops simultaneously. Absorbance measurement: Place the plate in a microplate reader and measure the absorbance (OD) of each well at 405 nm. When reading the absorbance, ensure that there are no bubbles or impurities in the wells of the plate to prevent inaccurate readings.

[0070] Test results: refer to Figure 6 , ALP expression levels in the CF / PEEK-MXene and CF / PEEK-MXene@Zn groups were significantly higher than those in the CF / PEEK group, and the differences were statistically significant (P<0.05). These results indicate that with prolonged osteogenic induction culture, ALP expression in all groups increased. The CF / PEEK-MXene@Zn group, containing the MXene / Zn coating, showed a significant advantage in promoting ALP expression, indicating that the MXene / Zn coating has a good synergistic promoting effect and can effectively enhance the composite scaffold's ability to support osteogenic differentiation.

[0071] Test Example 5 Test group: Same as Test Example 1.

[0072] Test method: The expression levels of key genes for osteogenic differentiation of rBMSCs were detected by real-time fluorescence quantitative PCR.

[0073] (1) Extraction of total RNA from cells: ① Discard the culture medium, add Trizol, and use a 1 mL pipette to repeatedly pipette for more than 30 times to ensure that there are no residual cell clusters (operate on ice to prevent RNA degradation), and then transfer to a 2 mL enzyme-free EP tube; ② Add 250 μL of chloroform to the EP tube, vortex and mix, and let it stand at room temperature for 5 minutes, then centrifuge at 4°C for 15 minutes; ③ Collect the supernatant layer, add an equal volume of isopropanol (250 μL), vortex and mix, let it stand at -20°C for 15 minutes, and then perform high-speed centrifugation at 4°C again (15 minutes); ④ Remove the supernatant, add 1 mL of 75% ethanol solution prepared with DEPC water to wash the precipitate, centrifuge under the same conditions for 15 minutes, and discard the supernatant; ⑤ Use a micropipette to remove the residual liquid, dry it in a clean room for 15 minutes, add 15 μL of DEPC water to dissolve the RNA precipitate, and incubate it in a metal bath at 55°C for 5 minutes. min to promote dissolution; ⑥ Use K2800 micro nucleic acid analyzer to determine RNA concentration and purity: after blank correction, take 1.5 μL sample for detection, and an A260 / A280 ratio of 1.8-2.1 is considered qualified.

[0074] (2) Reverse transcription reaction: ① Treat RNA with DNase I (Thermo, EN0521) (1 U / μg RNA, 37℃ / 30 min), and inactivate at 70℃ / 10 min after EDTA termination; ② Adjust to the same concentration with DEPC water (100-500 ng / μL is recommended) to prevent high concentration RNA from inhibiting reverse transcriptase activity; ③ Load the configured reverse transcription reaction system into a thermal cycler and perform the following thermal cycling parameters according to the preset program: initial denaturation at 25℃ for 10 min, cDNA synthesis at 42℃ for 15 min, and enzyme activity termination at 85℃ for 5 min. Immediately after the reaction is terminated, transfer the reaction tube to an ice bath for quenching to prevent the formation of nonspecific amplification products.

[0075] Test results: refer to Figure 7-Figure 9On day 14 of osteogenic differentiation, the expression of RUNX2 and ALP genes was significantly upregulated in the CF / PEEK-MXene and CF / PEEK-MXene@Zn groups compared to the CF / PEEK group (P < 0.05). Concurrently, the expression of the OCN gene also increased, but the difference was not statistically significant (P > 0.05). Furthermore, on day 14, the expression levels of osteogenic-related genes in the CF / PEEK-MXene@Zn group were slightly higher than those in the CF / PEEK-MXene group, but the difference between the two groups was also not significant. These results suggest that the CF / PEEK-MXene and CF / PEEK-MXene@Zn scaffolds can promote the expression of osteogenic-related genes in rBMSCs, especially in the middle and late stages of differentiation. This suggests that composite scaffolds containing MXene and Zn nanodots may have potential advantages in regulating the osteogenic differentiation of rBMSCs.

[0076] Test Example 6 Test group: Same as Test Example 1.

[0077] Test method: The CCK8 method was used to detect the cell proliferation data of HUVECs treated with different groups of composite biomimetic scaffold materials. The experimental steps were the same as those in Experimental Example 5.

[0078] Test results: refer to Figure 10 CCK-8 assay results showed that on day 1, there was no significant difference in cell proliferation among the CF / PEEK, CF / PEEK-MXene, and CF / PEEK-MXene@Zn groups (P>0.05). By day 4, the cell number in each group increased significantly, and the cell OD value of the CF / PEEK-MXene@Zn group was significantly higher than that of the CF / PEEK group (P<0.01). On day 7, the OD values of both the CF / PEEK-MXene and CF / PEEK-MXene@Zn groups were significantly higher than those of the CF / PEEK group (P<0.05).

[0079] Test Example 7 Test group: Same as Test Example 1.

[0080] Test method: Real-time fluorescence quantitative PCR was used to detect the effects of different scaffold materials on the expression levels of angiogenesis-related genes (VEGF, HIF-1α, CD31) in HUVEC cells. The experimental procedures were the same as those in Experiment 5.

[0081] Test results: refer to Figure 11-13After 3 days of culture, both the CF / PEEK-MXene and CF / PEEK-MXene@Zn groups significantly upregulated the expression of these genes compared to the CF / PEEK control group. The difference in HIF-1α gene expression between the two experimental groups was statistically significant (P < 0.05), while the CF / PEEK-MXene group also showed a significant increase in CD31 gene expression (P < 0.05). Although the expression levels of VEGF and HIF-1α genes in the CF / PEEK-MXene@Zn group were slightly higher than those in the CF / PEEK-MXene group, the difference between the two groups did not reach statistical significance (P > 0.05). These results indicate that both CF / PEEK-MXene and CF / PEEK-MXene@Zn scaffolds can effectively upregulate the expression of angiogenesis-related genes in HUVEC cells, particularly demonstrating a significant effect in promoting HIF-1α gene expression. In addition, the CF / PEEK-MXene@Zn group showed slightly better upregulation of VEGF and HIF-1α genes than the CF / PEEK-MXene group due to the synergistic effect of Zn nanodots in its composite coating, which may further enhance its angiogenic potential.

[0082] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-restrictive in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be included therein.

[0083] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Preparation method of CF / PEEK modified with MXene / Zn nano-coating, characterized in that: The steps include: S1: CF / PEEK scaffold pretreatment The CF / PEEK scaffold was ultrasonically cleaned with deionized water and dried; S2: CF / PEEK scaffold surface sulfonation The CF / PEEK scaffold dried in step S1 was added to 90% concentrated sulfuric acid with continuous magnetic stirring. After 3 minutes, the scaffold was removed and immediately ultrasonically cleaned in deionized water and dried. S3: Construction of PEI-TA Adhesion Layer Dissolve PEI and TA in deionized water to a concentration of 40 mg / mL each. Add the dried material from step S2 to 15 mL of PEI solution, stir for 10 min, then add 15 mL of TA solution and allow to react fully. When no flocs are evident, adjust the pH of the mixed solution to 4.2 ± 0.5 and seal overnight. S4: Preparation of CF / PEEK-MXene Materials The material obtained in step S3 was taken out and rinsed with deionized water, dried, and then 40 mL of 0.25 mg / mL MXene solution was added. The mixture was placed in a 37°C constant temperature shaker for 24 h to obtain a CF / PEEK-MXene material and a MXene solution system. S5: Preparation of CF / PEEK-MXene@Zn materials 40 mL of deionized water was added to the MXene solution system in step S4 for dilution, 137.4 mg of zinc acetate was weighed and added to the mixed solution and stirred continuously for 1 h, and then 624 mg of thioacetamide was added and stirred for 10 min. After sufficient reaction, the mixed solution and the material were transferred to a hydrothermal autoclave and heated at 60 ° C for 4 h. After cooling, the material was taken out to obtain CF / PEEK modified with MXene / Zn nanocoating, that is, CF / PEEK-MXene@Zn material.

2. The method for preparing CF / PEEK modified with MXene / Zn nano-coating according to claim 1, wherein: The drying conditions in steps S1, S2, and S4 are: temperature 60-80°C, time 2-4h.

3. The method for preparing CF / PEEK modified with MXene / Zn nano-coating according to claim 1, wherein: In step S3, the mixed solution is titrated with 0.1 mol / L HCl solution to a pH of 4.2±0.

5.

4. The CF / PEEK-MXene@Zn material prepared by the method according to any one of claims 1 to 3, characterized in that: The CF / PEEK-MXene@Zn material forms a specific MXene / Zn nano-coating structure on the surface of CF / PEEK through the above preparation steps.

5. The CF / PEEK-MXene@Zn material according to claim 4, characterized in that The surface of the CF / PEEK-MXene@Zn material is formed with a layer-by-layer progressive MXene / Zn nanodot active coating, and has a multi-level bionic structure with gradient mechanical adaptation properties and angiogenesis-osteogenesis coupling activity.

6. Use of the CF / PEEK-MXene@Zn material as claimed in claim 5 in the repair of large load-bearing bone defects.

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