A bone regeneration and repair titanium alloy with the function of regulating macrophage aging and a preparation method and application thereof

Abstract

The application discloses a bone regeneration and repair titanium alloy with the function of regulating macrophage aging, and a preparation method and application thereof. The bone regeneration and repair titanium alloy comprises a titanium alloy base body and a porous structure attached to the titanium alloy base body. The porous structure presents a multi-level size distribution, including long strip-shaped grooves and larger cavities (50 mu m cavities). The morphology in the grooves shows a coral-like ligament and a socket-shaped channel (1 mu m) structure. This can provide space and sites for the growth and adsorption of osteoblasts. The application also proposes a novel method for preparing non-oxidized and nitrided coral-like porous structures on commercial titanium implants by combining a vapor phase alloying (VPA) method with a vapor phase dealloying (VPD) method. The application provides an effective way for realizing in-vivo bone integration and reconstruction.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and biomaterials technology, specifically relating to a titanium alloy for bone regeneration and repair with the function of regulating macrophage aging, its preparation method and application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Compromised bone healing is one of the most devastating complications after orthopedic surgery, severely impacting patients' quality of life. Currently, there is no approved effective treatment for nonunion, and revision surgery and long-term drug therapy remain the standard of care, posing a significant clinical challenge. When tissues are subjected to physical or toxic damage, damaged or dying cells initiate an inflammatory response.

[0004] Sufficient contact at the fracture site, a vibrant biological environment, and a stable biomechanical environment are fundamental conditions for fracture healing. Monocytes and macrophages, with their significant plasticity and dynamic reprogramming capabilities, play crucial regulatory roles at all stages of tissue repair. Specifically, the initiation of immune regulation facilitates the delivery of nutrients and cytokines to the injury site, activates endogenous tissue stem cells, enhances cellular plasticity, and regenerates damaged cells in the body tissue. However, in impaired bone healing in the elderly, the regenerative potential and differentiation capacity of resident stem cells decline, which is associated with immunosense senescence in macrophages. Senescence induces metabolic reprogramming in these cells, disrupting their key functions, including debris clearance, phenotypic plasticity, and wound healing. Therefore, age-related phenotypic remodeling of tissue-resident immune cells within the microenvironment drives the functional decline in stem cell regeneration capacity, revealing a mechanistic link between immunometabolic reprogramming and age-related tissue repair impairment. Therefore, an alternative or synergistic strategy involves revitalizing macrophage function to enhance the regenerative capacity of resident bone marrow mesenchymal stem cells (BMMSCs) and restore youthful structural and functional characteristics of bone. However, effective strategies remain to be explored.

[0005] Three-dimensional scaffolds with suitable porous properties (including geometry, pore distribution, and porosity) play a crucial role in achieving this goal. Besides mechanical strength, nutrient diffusion, cell migration and differentiation, vascularization, and tissue regeneration rates are all influenced by porosity. Porous titanium (p-Ti) materials have been developed to improve cell-implant interactions due to their excellent biocompatibility, corrosion resistance, and mechanical properties. Traditional pore-forming methods include powder metallurgy, injection molding, and additive manufacturing. These processing techniques are complex, costly, and only suitable for fabricating simple pore structures, hindering the modulation of biological systems' responses to contextualized stimuli. For example, previous studies have revealed that nerve cells tend to penetrate pores rather than enter concave structures. Furthermore, mesenchymal stem cells tend to undergo chondrogenesis within pores of 90–120 μm in size, while actin filaments and myosin filaments tend to aggregate on concave surfaces. Pro-inflammatory cells tend to be secreted in larger pores, while vascularization typically occurs in concave areas. In the context of bone regeneration, the influence of scaffold geometry is particularly significant. Throughout bone remodeling, infiltration of macrophages and other cells (which are crucial for promoting angiogenesis and osteoblast migration) can be observed in larger pores ranging from 100 to 800 μm.

[0006] In contrast, vapor phase dealloying (VPD) technology, which utilizes the differences in saturated vapor pressure of components to form porous structures, shows great promise. While VPD has been successfully used to prepare porous copper, nickel, cobalt, and manganese, its application to titanium faces challenges. Due to titanium's high affinity for oxygen and nitrogen, VPD introduces a series of new challenges, including the risk of oxidation and nitridation of porous structures. On the other hand, most studies focus on pore sizes ranging from several micrometers to 100 μm to assess the impact of scaffold pore size on host immune responses. The preparation of titanium alloys with specific pore sizes may have beneficial effects on regulating the bone immune microenvironment. However, a comprehensive understanding of how these processes are coordinated during tissue repair and how age-related inflammatory dysregulation affects them at the organ, tissue, and cellular levels remains lacking. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a titanium alloy for bone regeneration and repair with the function of regulating macrophage aging, as well as its preparation method and application.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a bone regeneration and repair titanium alloy with the function of regulating macrophage aging. The bone regeneration and repair titanium alloy comprises a titanium alloy matrix and a porous structure attached to the titanium alloy matrix. The porous structure exhibits a multi-level size distribution, including elongated grooves (100-150 μm wide) and larger pores (30-50 μm). The morphology within the grooves displays coral-like ligaments and nest-like channels (0.5-2 μm). This provides space and sites for osteoblast growth and adsorption.

[0010] This invention also proposes a novel method for preparing an oxidation- and nitriding-free coral-like porous structure on commercial titanium implants by combining vapor phase alloying (VPA) with vapor phase dealloying (VPD).

[0011] Secondly, the present invention provides a method for preparing a bone regeneration and repair titanium alloy with the function of regulating macrophage aging, comprising the following steps: S1, the pretreatment of titanium alloys, including grinding and solvent cleaning, and annealing in a tube furnace at 500~700℃ for 1~3 hours to relieve stress; to avoid deformation of thin-walled areas after vapor phase treatment, deviating from the original shape; S2, Vapor phase alloying treatment of titanium alloy: The titanium alloy pretreated in step S1 is sealed together with zinc particles in a container under vacuum, and vapor phase alloying (VPA) is carried out at 600~800℃ for 1~15 hours to obtain the precursor. In step S2, the container is a quartz tube with a vacuum level of 1~100 Pa.

[0012] In step S2, vapor phase alloying is performed in a muffle furnace.

[0013] S3, Vapor phase dealloying treatment: Place the titanium alloy precursor after the vapor phase alloying treatment in step S2 in a container and incubate at 550~750℃ for 1×10⁻⁶ days. -2 Vapor phase dealloying (VPD) is performed by holding the material at ~100 Pa for 1 to 2 hours.

[0014] In step S3, vapor-phase dealloying is performed in a tube furnace.

[0015] Furthermore, in steps S2 and S3, titanium foil is placed on both sides of the titanium alloy in the container to remove excess air. The titanium foil on both sides can react with the trace amounts of oxygen, nitrogen and other gases remaining in the vacuum environment to eliminate the influence on the sample. Based on the titanium foil combined with the vacuum environment, an oxygen-free environment and the generation of nitrogen-free products are achieved.

[0016] Furthermore, in step S3, an argon-hydrogen mixture containing 7 vol.% hydrogen is introduced into the tube furnace to further prevent sample oxidation during the dealloying process.

[0017] Furthermore, this invention delves into the complexity of the prepared hierarchical porous ligament structure, examining its formation process and evaluating its impact on osteogenic properties. First, thanks to its coral-like hierarchical structure with niche-like pores, the porous titanium alloy (p-Ti) exhibits excellent osteogenic properties, promoting osteogenic differentiation and expression of bone marrow mesenchymal stem cells (BMMSCs). Second, the bone immunomodulatory effects of p-Ti were further investigated in vitro by co-culturing p-Ti with macrophages. The coral-like hierarchical structure of p-Ti can promote ARG1... + Macrophage production. Furthermore, by establishing dual-validation bone defect models in aging rats and rabbits, the in vivo osseointegration and remodeling effects of p-Ti implants were further investigated.

[0018] Based on the above research findings, as a third aspect, this invention provides the application of the aforementioned titanium alloy for bone regeneration and repair with macrophage-regulating aging effects in the preparation of bone implants.

[0019] In the third aspect of the application, the bone regeneration and repair titanium alloy with the function of regulating macrophage aging has the following characteristics: (1) Promotes osteogenic differentiation and expression of bone marrow mesenchymal stem cells (BMMSCs); (2) Promote ARG1 + The production of macrophages; (3) Achieve in vivo bone integration and reconstruction.

[0020] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: (1) This invention provides a titanium alloy for bone regeneration and repair with the function of regulating macrophage aging, which promotes osteogenic differentiation and expression of bone marrow mesenchymal stem cells (BMMSCs) and promotes ARG1. + The production of macrophages enables bone integration and reconstruction within the body.

[0021] (2) This invention provides a method for preparing a titanium alloy for bone regeneration and repair with the function of regulating macrophage aging. Compared with other technologies, this technology can firstly directly process existing implant workpieces, etc., without the need to redesign methods such as powder sintering and 3D printing; secondly, this method can directly form multi-level pores on the surface, which is conducive to the adsorption of different cells and cell tails.

[0022] (3) The bone regeneration and repair titanium alloy provided by the present invention, which has the function of regulating macrophage aging, can effectively promote osteogenic differentiation and reverse macrophage aging in vitro, and has great potential to promote the regeneration of aging bone, providing an effective means for the treatment of fractures, especially for the treatment of bone healing disorders in elderly patients. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 This is a characterization of the titanium raw material and porous titanium implant in Example 1; wherein, (a) a schematic diagram of the porous titanium preparation process, (b) a photograph of the p-Ti disk after VPD treatment, (c) the surface morphology of porous titanium, (d) the magnified morphology of porous titanium in the yellow area of ​​(c), (e) elemental content, (f) Zn release amount, (g) corrosion resistance comparison, and (h) X-ray diffraction pattern.

[0025] Figure 2 The effect of porous titanium prepared for Example 1 on osteogenic differentiation of rat BMMSCs; wherein, (ad) expression of ALP (alkaline phosphatase), OCN (osteocalcin), RUNX2 (Runt-related transcription factor 2) and COL1 (type I collagen) in rat BMMSCs in each implant group on day 2 of culture; (e) quantitative analysis by immunofluorescence staining: expression level of ALP in rat BMMSCs in each implant group on day 3 of culture; (f) immunofluorescence staining images: expression of ALP in rat BMMSCs in each implant group on day 3 of culture, with scale bars of 50 μm in the figures. This indicates that compared to the titanium group, P < 0.05. This indicates that P < 0.01 compared to the titanium group.

[0026] Figure 3 The porous titanium prepared in Example 1 was used to regulate macrophage immune senescence and inhibit the secretion of inflammatory factors. Specifically, (ad) RT-PCR was used to detect the expression of iNOS, CD206, ARG1, and BMP2 in macrophages cultured on the porous titanium surface; (e) representative images of immunofluorescence staining of macrophage markers (iNOS and CD206) on the porous titanium surface; (fg) ELISA was used to detect the secretion levels of IL10 and IL6 in macrophages on the porous titanium surface on day 2 of culture; (h) Western blot quantitative analysis results; and (i) Western blot was used to detect the expression of CD163, IL1β, p16, and p21 in macrophages in the conditioned medium system. This indicates that compared to the titanium group, P < 0.05. This indicates that P < 0.01 compared to the titanium group.

[0027] Figure 4 The porous titanium implant prepared in Example 1 promotes osseointegration in a bone defect model; (a) actual image of the femoral specimen, (b) image of hard tissue sections stained with calcein / alizarin red double staining, where the scale bar is 200 μm, (c) quantitative analysis of femoral length in each group 8 weeks after implantation, (d) quantitative analysis of calcein-stained hard tissue sections, (e) representative H&E staining images of rat organs in different treatment groups, where the scale bar is 500 μm, (fi) quantitative analysis of bone volume fraction (BV / TV%), number of trabeculae (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th), and (j) Micro-CT coronal, sagittal, three-dimensional reconstruction, and new bone imaging, where the scale bar in the Micro-CT coronal and sagittal images is 5 mm, and the scale bar in the three-dimensional reconstruction and new bone imaging images is 2 mm. This indicates that compared to the titanium group, P < 0.05. This indicates that P < 0.01 compared to the titanium group.

[0028] Figure 5 The images show the morphology and composition of the titanium alloy prepared in Example 1; where a is the low-magnification SEM morphology, b is the high-magnification SEM morphology, c is the energy dispersive spectroscopy (EDS) curve, and d is the content of each element as determined by EDS.

[0029] Figure 6 The images show the morphology of the titanium alloy prepared in Comparative Example 1; where a is the low-magnification SEM morphology and b is the high-magnification SEM morphology.

[0030] Figure 7 The images show the morphology and composition of the titanium alloy prepared in Example 2; where a is the low-magnification SEM morphology, b is the high-magnification SEM morphology, c is the energy dispersive spectroscopy (EDS) curve, and d is the content of each element as measured by EDS. Detailed Implementation

[0031] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] The increasing global aging population has made age-related bone diseases, driven by progressive loss of bone homeostasis and decreased regenerative capacity, a key frontier area of ​​modern healthcare challenges. Compromised bone healing remains one of the most serious complications after orthopedic surgery, posing a significant clinical challenge. It is characterized by chronic low-grade inflammation (inflammatory senescence), marked by the persistent activation of inflammatory pathways. This process is accompanied by a decline in immune system function, particularly in the macrophage population that coordinates inflammatory homeostasis. As key regulators of tissue repair, immune regulation plays a central role in delivering nutrients and cytokines to the injury site, activating endogenous tissue stem cells to enhance cellular plasticity, and regenerating damaged cells in tissues. However, in the context of bone defects, resident stem cells exhibit diminished regenerative potential and reduced differentiation capacity, a process associated with macrophage immunosenescence.

[0033] The present invention will be further described below with reference to the embodiments.

[0034] Example 1 Commercial titanium rods (CP-Ti, grade 2) of different diameters were cut into Φ10 mm × 2 mm (titanium disks) and Φ2 mm × 5 mm (titanium rods) using a diamond wire cutter. The titanium disks served as characterization substrates in in vitro studies and as base materials for in vivo subcutaneous ectopic osteogenic experiments, while the titanium rods were used for in vivo femoral implantation experiments. All samples were sequentially polished with P400, P800, and P1200 silicon carbide sandpaper, followed by ultrasonic cleaning in deionized water, alcohol, and acetone, respectively. The cleaned titanium disks and rods were annealed in a tube furnace at 700°C for 3 hours to relieve stress. Subsequently, the annealed titanium disks or rods were sealed together with zinc particles in a quartz tube (within a low vacuum of 1–10 Pa) and subjected to vapor phase alloying (VPA) at 700°C for 6 hours in a muffle furnace. Finally, the alloyed titanium disk or titanium rod precursor is placed in a quartz tube and placed in a tube furnace at 650°C. In the tube furnace, titanium foil is placed at both ends of the quartz tube to remove trace amounts of oxygen and nitrogen remaining in the vacuum environment. An argon-hydrogen mixture containing 7 vol.% hydrogen is used as the carrier gas to further ensure that the sample is not oxidized. The sample is held at 100 Pa for 2 hours for vapor phase dealloying (VPD) treatment to obtain a porous titanium disk (p-disk) or porous titanium rod (p-rod). The preparation process is as follows: Figure 1 As shown in Figure a.

[0035] Example 2 The same preparation process as in Example 1 was used, except that the temperature was kept at 700°C for 6 hours and at VPD: 650°C and 100 Pa for 2.5 hours.

[0036] Comparative Example 1 The same preparation process as in Example 1 was used, except that VPA was kept at 700°C for 15 hours and VPD was kept at 650°C and 100 Pa for 2 hours.

[0037] Experimental Example 1 1. Characterization of porous titanium alloys The actual photographs and microstructures of the p-Ti disk prepared in Example 1 are shown below. Figure 1 As shown in Figure 2, the actual photograph reveals a distinct crack-like texture. Unlike existing technologies that primarily feature single-pore structures, the porous titanium alloy prepared in this application exhibits a hierarchical porous structure under scanning electron microscopy (SEM). At low magnification, it displays elongated ravines (100 μm ravines) and larger pores (50 μm cavity). Further magnification of the morphology within the ravines reveals coral-like ligaments and nest-like channels (1 μm). Energy dispersive spectroscopy (EDS) results show the presence of a small amount of Zn (1.6 at.%). Figure 1 (e) It was not removed during the vapor-phase dealloying process. For example... Figure 1 Inductively coupled plasma mass spectrometry (ICP-MS) results showed that residual Zn was slowly released in PBS solution, promoting osteoblast growth. The corrosion resistance of the porous material was tested using a Tafel assay. Figure 1 (medium g), compared with commercial Ti (-0.177 V vs. saturated calomel electrode, 3.932 × 10⁻⁶ V), and commercial Ti (-0.177 V vs. saturated calomel electrode, 3.932 × 10⁻⁶ V). -6 A) Compared to a saturated calomel electrode, the p-Ti disk has a more positive corrosion potential (-0.142 V vs. saturated calomel electrode) but a slightly higher corrosion current (5.018 × 10⁻⁶ V). -6 A) indicates that p-Ti disks have better corrosion resistance, but the continuous release of residual zinc from the porous structure increases the self-corrosion current. Figure 1 (f). X-ray diffraction results show that the material is dominated by α-phase Ti with a small amount of β-phase Ti, which may be due to the phase transformation caused by the Zn extraction process. Figure 1 (h).

[0038] 2. Effects of porous titanium on osteogenic differentiation of rat BMMSCs The effect of the porous titanium (p-Ti) scaffold prepared in Example 1 on the osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMMSCs) was evaluated using RT-PCR. Rat BMMSCs (4 × 10⁻⁶) were used in the study. 4After culturing the scaffold surface in 24-well plates for 3 days, the expression levels of osteogenic-related genes, including alkaline phosphatase (ALP), osteocalcin (OCN), Runt-associated transcription factor 2 (RUNX2), and type I collagen (COL1), were quantitatively analyzed by reverse transcription polymerase chain reaction (RT-PCR). Compared with the titanium group (commercial titanium without porous treatment), the porous titanium scaffold significantly increased the mRNA expression levels of ALP, OCN, RUNX2, and COL1. Figure 2 (ad), with the expression level of the OCN gene increasing nearly fourfold.

[0039] Immunofluorescence staining was used to detect ALP protein expression, and the results showed that ALP in the porous titanium group ( Figure 2 The expression levels of e and f in the middle group were significantly higher than those in the titanium group.

[0040] 3. Porous titanium regulates macrophage immune aging and inhibits the secretion of inflammatory factors. Macrophages were further seeded onto the surface of porous titanium modules and titanium implants in six-well plates and cultured for 2 days. Figure 3 As shown in the figure below, compared with other groups, the expression of mannose receptor (CD206) in macrophages was significantly increased in the porous titanium group (P<0.05), while the expression of inducible nitric oxide synthase (iNOS) showed no statistically significant difference. Meanwhile, the expression of arginase 1 (ARG1) and bone morphogenetic protein 2 (BMP2), which are related to M2 polarization and osteogenic processes, was significantly increased in the porous titanium group (P<0.01), an effect attributed to its porous structure. Previous studies have found that ARG1... + Macrophage subsets are activated in young tissues but suppressed in senescent tissues; these subsets are key regulators of aging-related regenerative immune responses. Macrophage senescence leads to functional impairment and may trigger the accumulation of abnormal proteins (pathological markers of various diseases). M2 macrophages participate in immune regulation by secreting anti-inflammatory factors such as IL-4 and TGFβ, and are highly expressed in young individuals. Immunofluorescence staining showed that the density of CD206+ cells in the porous titanium group was significantly higher than that in the titanium group (…). Figure 3 The study confirmed its significant tendency to induce M2 polarization. Enzyme-linked immunosorbent assay (ELISA) showed that the porous titanium group promoted the secretion of the M2-related cytokine IL10 (…). Figure 3 f, P<0.05), while inhibiting the release of pro-inflammatory factor IL6 (P<0.05). Figure 3 (g, P<0.01). The above data indicate that porous titanium promotes bone regeneration by regulating the M2 immunosenescence phenotype (enhanced IL10 secretion).

[0041] The porous titanium prepared in Example 1 possesses a high specific surface area and a three-dimensional porous structure, comprising micropores of approximately 1 μm, cavities of approximately 50 μm, and ravines approximately 100 μm wide. This structure effectively promotes ARG1. + Macrophage generation and M2 polarization. Experiments showed that porous titanium significantly increased the expression of M2 polarization-related proteins and induced macrophages to transform into an anti-aging phenotype, while significantly reducing the expression of aging markers p16 and p21 in these immunomodulatory cells. Figure 3 (h in the middle, i in the middle). This phenotypic regulation may play a key role in promoting tissue repair and regeneration.

[0042] The above results indicate that porous titanium can effectively promote osteogenic differentiation, reverse macrophage senescence, and has great potential to promote the regeneration of senescent bone in vitro.

[0043] 4. Porous titanium implants promote osseointegration in bone defect models To evaluate the in vivo osteogenic effects of titanium and porous titanium implants, cylindrical implants (titanium rods and porous titanium rods) were implanted into the distal femoral metaphysis of rats with bone defects. The entire surgical procedure adhered to aseptic techniques and the ethical standards of Qilu Hospital of Shandong University. The specific procedures included femoral exposure, implantation site localization, drilling, and implantation. Calcein Green was administered intraperitoneally 4 weeks postoperatively, Alizarin Red was administered at 6 weeks, and a micro-CT scan was performed at 8 weeks.

[0044] Methodology: The rat femoral defect model construction and in vivo experimental protocol were approved by the Animal Nursing Committee of Qilu Hospital, Shandong University (Approval No.: DWLL-2021-034), and all procedures followed the guidelines of the hospital's Ethics Committee. Rats were anesthetized under aseptic conditions, and a bone defect model was constructed in the distal femur. A cylindrical implant (Φ2 mm × 5 mm) was then implanted into the defect site to observe in vivo bone regeneration and repair. At weeks 4 and 6 post-surgery, early and late-stage new bone were labeled with Calcein Green and Alizarin Red, respectively, via intraperitoneal injection. At week 8, rats were euthanized, and femoral tissue was harvested. Changes in femoral length after implantation were assessed by measuring the proximal-distal femoral distance.

[0045] Results: Femoral length measurements 8 weeks after implantation showed no significant difference between the titanium group and the porous titanium group. Figure 4 (a) and (c) in section 4 confirmed that porous titanium implantation does not affect bone development. Laser confocal imaging of bone tissue sections showed that in dual-labeling detection of calcein (early osteogenic marker) and alizarin red (late osteogenic marker), the porous titanium group had a significant advantage in both early and late osteogenic stages. Figure 4(b in group B, d in group 4) Further comparison of the pathological findings of rat organs in each experimental group confirmed that the physiological state of solid organs was not affected by the implant. Figure 4 (e), which verified its biocompatibility in vivo.

[0046] Micro-CT three-dimensional reconstruction confirmed the formation of a dense new bone layer around the porous titanium implant: bone volume fraction (BV / TV) increased by 50%, trabecular bone number (Tb.N) and thickness (Tb.Th) significantly increased, and trabecular bone separation (Tb.Sp) decreased. Figure 4 In the middle section, a three-dimensional perspective more intuitively displays the uniform and dense new bone structure surrounding the porous titanium assembly. Figure 4 (j).

[0047] These results confirm that porous titanium implants can effectively increase early and late-stage osteogenic capacity while ensuring normal bone development, providing a breakthrough solution for the clinical treatment of bone defects.

[0048] Experimental Example 2 Figures 5-7 The images show the morphology of the titanium alloy products prepared in Examples 1, 2, and 1, including low-magnification and high-magnification SEM images of the titanium alloy product prepared in Example 1. Figure 5 (a and b), energy spectrum curves ( Figure 5 (c) and element content diagram ( Figure 5 (d) Low-magnification and high-magnification SEM images of the titanium alloy product prepared in Comparative Example 1 ( Figure 6 (a and b), low-magnification and high-magnification SEM images of the titanium alloy product prepared in Example 2 ( Figure 7 (a and b), energy spectrum curves ( Figure 7 (c) and element content diagram ( Figure 7 As shown in d), the VPA-VPD process within the specified range can achieve a dual-continuous ligament and pore structure. Excessive VPA process time (15 hours) leads to an excessively thick precursor layer, and the dealloying process generates significant geometric stress, causing the porous layer to detach. Extending the dealloying time can effectively reduce the zinc content in the porous layer.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a titanium alloy for bone regeneration and repair with the function of regulating macrophage aging, characterized in that, Includes the following steps: S1, the pretreatment of titanium alloys, including grinding and solvent cleaning, and annealing in a tube furnace at 500~700℃ for 1~3 hours to relieve stress; S2, Vapor phase alloying treatment of titanium alloy: The titanium alloy pretreated in step S1 is sealed together with zinc particles in a container under vacuum, and vapor phase alloying is carried out at 600~800℃ for 1~15 hours to obtain the precursor. S3, Vapor phase dealloying treatment: Place the titanium alloy precursor after the vapor phase alloying treatment in step S2 in a container and incubate at 550~750℃ for 1×10⁻⁶ days. -2 Vapor phase dealloying is performed by holding the temperature at ~100 Pa for 1~2 hours. In step S2, the container is a quartz tube with a vacuum level of 1~100 Pa. In steps S2 and S3, titanium foil is placed on both sides of the titanium alloy in the container to remove excess air. In step S3, an argon-hydrogen mixture containing 7 vol.% hydrogen is introduced into the tube furnace.

2. The method for preparing the bone regeneration and repair titanium alloy with macrophage senescence regulating effect according to claim 1, characterized in that, In step S2, vapor phase alloying is performed in a muffle furnace.

3. The method for preparing the bone regeneration and repair titanium alloy with macrophage senescence regulating effect according to claim 2, characterized in that, In step S3, vapor-phase dealloying is performed in a tube furnace.

4. The bone regeneration and repair titanium alloy with macrophage-regulating aging effects prepared by the method of any one of claims 1 to 3, characterized in that, The bone regeneration and repair titanium alloy comprises a titanium alloy matrix and a porous structure attached to the titanium alloy matrix. The porous structure exhibits a multi-level size distribution, including elongated grooves and pores. The morphology within the grooves shows coral-like ligaments and nest-like channel structures. The elongated grooves are 100-150 μm wide, the pores are 30-50 μm in size, and the hollow channels are 0.5-2 μm in size.

5. The application of the titanium alloy for bone regeneration and repair with macrophage senescence regulation as described in claim 4 in the preparation of bone implants.

6. The application according to claim 5, characterized in that: The bone regeneration and repair titanium alloy with the function of regulating macrophage aging has the following characteristics: (1) Promotes osteogenic differentiation and expression of bone marrow mesenchymal stem cells; (2) Promote ARG1 + The production of macrophages; (3) Achieve in vivo bone integration and reconstruction.

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