A porous titanium alloy support rod and a preparation method and application thereof

Abstract

The application relates to the technical field of bone grafting, and particularly discloses a porous titanium alloy support rod as well as a preparation method and application thereof. The porous titanium alloy support rod comprises a head body part and a tail part which are fixedly connected, the head body part is a reticular porous cylindrical shape, the head body part is composed of a plurality of hexagonal honeycomb structure units, and the tail part is a solid cylindrical body; the average pore diameter of the porous titanium alloy support rod is 700 microns to 800 microns, the porosity is 70% to 75%, and the average wire diameter is 340 microns to 400 microns. The porous titanium alloy support rod provided by the application has a bearing capacity as high as 713 N, can be effectively integrated with in-vivo bone tissue, can promote the bone formation of the support, and can increase the bone formation amount.

Description

Technical Field

[0001] This invention relates to the field of bone transplantation technology, specifically to a porous titanium alloy support rod, its preparation method, and its application. Background Technology

[0002] Currently, avascular necrosis of the femoral head (ONFH) remains a common, refractory, and progressive disease faced by orthopedic surgeons. The primary cause is damage or interruption of blood supply to the femoral head due to various reasons, leading to tissue necrosis and structural destruction within the femoral head, resulting in increased internal pressure and consequently pain and functional impairment. Early symptoms of ONFH are often subtle, and the disease progresses rapidly. Without timely treatment, it can easily progress to femoral head collapse or secondary hip arthritis. However, most patients do not respond well to non-surgical treatment and may ultimately have to undergo total hip replacement (THA). Most young patients are unwilling to undergo THA. Therefore, preserving the patient's own hip joint and delaying or even avoiding THA as much as possible is a major challenge for both clinicians and ONFH patients. Current surgical interventions for early and mid-stage ONFH include: single-port or multi-port core decompression, osteotomy, core decompression combined with vascularized or non-vascularized bone grafting, core decompression combined with bio-assisted therapy (such as concentrated stem cells), and core decompression combined with tantalum rod implantation. Each of these treatments has its advantages and disadvantages, but core decompression remains a commonly used surgical method for treating the pre-collapse stage of ONFH. Therefore, researchers are constantly seeking new support materials to provide channel mechanical support and promote bone repair.

[0003] 3D-printed porous titanium alloy support rods have demonstrated therapeutic efficacy in large animal experiments for treating early-stage osteoarthritis of the femoral head (ONFH). However, existing support rods with porous structures have traditional rectangular structural units within their internal microstructure. These rods exhibit low yield strength and poor load-bearing capacity, resulting in slow bone ingrowth in the later stages of in vivo repair. This slow ingrowth cannot match the rate of local bone resorption in ONFH, leading to subchondral bone collapse in the femoral head region. Therefore, a novel porous titanium alloy support rod is urgently needed. Summary of the Invention

[0004] To develop a porous titanium alloy support rod that enhances bone and blood vessel regeneration capabilities, this invention provides a porous titanium alloy support rod, its preparation method, and its applications. The porous titanium alloy support rod provided by this invention has a load-bearing capacity of up to 713 N, can effectively integrate with bone tissue in vivo, promotes scaffold osteoogenesis, and increases bone formation.

[0005] This invention provides a porous titanium alloy support rod, which includes a head and a tail portion fixedly connected. The head portion is a mesh-like porous cylinder composed of several hexagonal honeycomb structural units, and the tail portion is a solid cylinder. The porous titanium alloy support rod has an average pore diameter of 700μm to 800μm, a porosity of 70% to 75%, and an average wire diameter of 340μm to 400μm.

[0006] This invention sets the internal structure of a porous titanium alloy support rod as a hexagonal honeycomb structure unit, and limits the average pore diameter of the support rod to 700μm to 800μm, the porosity to 70% to 75%, and the average wire diameter to 340μm to 400μm. Compared with the traditional rectangular structure unit, this invention significantly improves the load-bearing capacity of the porous titanium alloy support rod. The load-bearing capacity of the porous titanium alloy support rod of this invention is 713N. The porous titanium alloy support rod provided by this invention can effectively integrate with bone tissue in vivo and promote osteogenesis.

[0007] This invention also provides a method for preparing the porous titanium alloy support rod. The porous titanium alloy support rod structure is designed using CAD software, and then printed using selective laser melting technology. The printing parameters are as follows: upper skin laser power 80W~100W, scanning speed 1000mm / s~1300mm / s; inner filling laser power 300W~400W, scanning speed 800mm / s~1000mm / s; lower skin laser power 40W~60W, scanning speed 1200mm / s~1400mm / s. After printing, the rod is cleaned with an ultrasonic cleaner, dried, and then sterilized by Co60 radiation for later use.

[0008] The present invention also provides a barium titanate coated porous titanium alloy support rod, wherein the barium titanate coated porous titanium alloy support rod is obtained by coating the surface of the porous titanium alloy support rod with a barium titanate coating.

[0009] This invention also provides a method for preparing the porous titanium alloy support rod with barium titanate coating. A clear electrolyte is obtained by mixing ammonium fluoride, deionized water, and ethylene glycol in a ratio of 0.3714 g to 0.4 g: 5 mL: 200 mL. Using a platinum sheet as the cathode and the porous titanium alloy support rod as the anode, anodizing is performed. After cleaning and drying, the temperature is increased to 400°C at a rate of 0.5°C to 1.5°C / min and held for 2.5 to 3.5 hours, followed by heat treatment. The heat-treated porous titanium alloy support rod is then immersed in a barium hydroxide solution and heated to 200°C to 220°C, held for 1.5 to 2.5 hours, and then cooled to room temperature at a rate of 0.5°C to 1.5°C / min. The rod is then polarized at a voltage of 10 kV to 13 kV for 30 minutes. After polarization, the rod is cleaned, dried, and sterilized by Co60 radiation to obtain the porous titanium alloy support rod with barium titanate coating.

[0010] The present invention also provides the application of the porous titanium alloy support rod or the porous titanium alloy support rod with barium titanate coating in the preparation of bone graft materials.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: The hexagonal honeycomb porous titanium alloy support rod prepared in Example 1 of this invention exhibits the highest yield strength, optimal mechanical properties, and an average load-bearing capacity of 713 N. It also demonstrates superior mechanical load-bearing performance and better in vivo osseointegration and ingrowth effects. Applying a barium titanate coating to the surface of the porous titanium alloy support rod prepared in this invention significantly improves bone volume fraction, trabecular bone thickness, and bone mineral density. The barium titanate-coated porous titanium alloy support rod can generate an effective force-electric conversion effect in vivo, thereby promoting osteogenesis. This support rod material holds promise for addressing the problems of decreased femoral head mechanical properties and difficulties in osteogenic repair after ONFH core decompression surgery. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 The figure shows a schematic diagram of the porous titanium alloy support rod designed in Embodiment 1 of the present invention; in the figure, A is a front view of the porous titanium alloy support rod designed in Embodiment 1; B is a three-dimensional structural schematic diagram of the porous titanium alloy support rod; C is a schematic diagram of the head structure of the porous titanium alloy support rod; D is a schematic diagram of the bottom surface structure of the tail of the porous titanium alloy support rod; and E is a schematic diagram of the cross-sectional structure of the body of the porous titanium alloy support rod. Figure 2 The diagram shows the porous titanium alloy support rod structure designed in Comparative Example 1 of this invention; in the diagram, A is a schematic diagram of the overall structure of the porous titanium alloy support rod; B is a schematic diagram of the head structure of A; C is a schematic diagram of the tail bottom structure of A; and D is a schematic diagram of the cross-sectional structure of the body. Figure 3 The diagram shows the porous titanium alloy support rod structure designed in Comparative Example 2 of this invention. In the diagram, A is a schematic diagram of the overall structure of the porous titanium alloy support rod; B is a schematic diagram of the head structure of A; C is a schematic diagram of the tail bottom structure of A; and D is a schematic diagram of the cross-sectional structure of the body of A. Figure 4The figures show the physical images of the porous titanium alloy support rods, discs, and body supports prepared in Examples 1 and Comparative Examples 1-2. In the figures, A, from top to bottom, shows the porous titanium alloy support rods prepared in Comparative Examples 1, 2, and 1; B, from left to right, shows the porous titanium alloy support rods prepared in Comparative Examples 1, 2, and 1; C is a physical image of the disc prepared in Comparative Example 1; D is a physical image of the disc prepared in Comparative Example 2; E is a physical image of the disc prepared in Example 1; F is a physical image of the body support structure prepared in Comparative Example 1; G is a physical image of the body support structure prepared in Comparative Example 2; and H is a physical image of the body support structure prepared in Example 1. Figure 5 The figure shows the in vitro mechanical performance evaluation of the porous titanium alloy support rod. In the figure, A is a picture of the self-made fixture and indenter used for testing in this invention; B is a picture of the porous titanium alloy support rod after in vitro mechanical performance evaluation, which shows the yield strength test made to simulate the pressure effect inside the femoral head. a, b, c, d and e are the states of slight deformation, slight fracture, partial fracture and complete breakage, respectively. Figure 6 The figures show the experimental force-displacement curves of the maximum load-bearing capacity of the porous titanium alloy support rod prepared according to the present invention. In the figures, A is the experimental force-displacement curve of the maximum load-bearing capacity of the porous titanium alloy support rod prepared in Comparative Example 1, wherein Attachments 1-5 are all duplicate samples of the porous titanium alloy support rod; B is the experimental force-displacement curve of the maximum load-bearing capacity of the porous titanium alloy support rod prepared in Comparative Example 2, wherein Attachments 1-5 are all duplicate samples of the porous titanium alloy support rod; C is the experimental force-displacement curve of the maximum load-bearing capacity of the porous titanium alloy support rod prepared in Example 1, wherein Attachments 1-5 are all duplicate samples of the porous titanium alloy support rod. Figure 7 The maximum load-bearing capacity statistical diagram (A) and absorbance value comparison diagram (B) of the porous titanium alloy support rods prepared in Example 1 and Comparative Examples 1-2 are shown. Figure 8 The effects of the porous titanium alloy support rod of the present invention on bone volume fraction, trabecular bone thickness, and bone mineral density were detected by Micro-CT. In the figure, A represents the bone volume fraction of the different treatment groups at week 6 after stent implantation; B represents the trabecular bone thickness of the different treatment groups at week 6 after stent implantation; C represents the bone mineral density of the different treatment groups at week 6 after stent implantation; D represents the bone volume fraction of the different treatment groups at week 12 after stent implantation; E represents the trabecular bone thickness of the different treatment groups at week 12 after stent implantation; and F represents the bone mineral density of the different treatment groups at week 12 after stent implantation. Figure 9The figures show histological sections and VG staining results of scaffolds prepared in Example 1 and Comparative Examples 1-2 after 6 and 12 weeks of implantation; black represents the scaffolds, and red represents collagen fibers; in the figures, A is the histological section and VG staining result of the scaffold prepared in Comparative Example 1 after 6 weeks of implantation; B is the histological section and VG staining result of the scaffold prepared in Comparative Example 2 after 6 weeks of implantation; C is the histological section and VG staining result of the scaffold prepared in Example 1 after 6 weeks of implantation; D is the histological section and VG staining result of the scaffold prepared in Comparative Example 1 after 12 weeks of implantation; E is the histological section and VG staining result of the scaffold prepared in Comparative Example 2 after 12 weeks of implantation; and F is the histological section and VG staining result of the scaffold prepared in Example 1 after 12 weeks of implantation. Figure 10 Macroscopic images and microscopic surface images of the PT group discs and the PTB group discs coated with barium titanate are shown. In the figures, A is the macroscopic image of the PT group discs; B is the macroscopic image of the PTB group discs; C is the surface of the PT group discs observed by SEM (20000x); and D is the surface of the PTB group discs observed by SEM (20000x). Figure 11 The energy dispersive spectroscopy (EDS) diagrams show the elemental composition and content of the PT group discs and the PTB group discs coated with barium titanate. In the diagrams, A represents the elemental composition and content of the PT group, and B represents the elemental composition and content of the PTB group. Figure 12 The graph shows the piezoelectric coefficient (d33) of two sets of discs, n=9, **** P<0.0001. Figure 13 SEM observation of cells (blue) and discs (gray) after in vitro co-culture with load; in the figure, A and B represent SEM images of cell-pure titanium alloy disc co-culture, respectively, A is 2500x (2500x); B is 1000x (1000x); C and D are SEM images of cell-barium titanate coated disc co-culture, C is 2500x (2500x); D is 1500x (1500x). Figure 14 The effect of barium titanate coated porous titanium alloy on cell apoptosis is shown in the figure. In the figure, A, B and C represent the flow cytometry results of the PT group, PTB group and blank control group, respectively. D is a statistical graph of the apoptosis rate of cells after treatment of PTB group, PT group and blank control group. Figure 15 Calcein-AM / PI staining was performed on two groups of cells after co-culturing with discs (n=5). * P<0.05; In the figure, C and F are fluorescence fusion images of A and B, and D and E, respectively; G, H, and I are statistical comparisons of the average fluorescence intensity of Calcein-AM, PI, and Merge in the two groups, respectively. Figure 16The figures show the osteoclast induction results after co-culture with load cells; in the figures, A, B, and C are 100x light micrographs of the PT group (blue arrows indicate osteoclasts); D, E, and F are 100x light micrographs of the PTB group (blue arrows indicate osteoclasts); G is a comparison of the total number of cells under the microscope between the two groups, **** P < 0.0001; H is a statistical graph of the osteoclast count under the microscope between the two groups, ** P < 0.01; I is a statistical graph of the percentage of osteoclasts in the two groups, * P < 0.05. Figure 17 Intraoperative and postoperative X-ray images of rabbit femoral head core decompression and support rod implantation: In the figure, A is the intraoperative anteroposterior X-ray (blue arrow indicates the positioning pin); B is the intraoperative lateral X-ray (blue arrow indicates the positioning pin); C is the postoperative anteroposterior X-ray (blue arrow indicates the implanted support rod); D is the postoperative lateral X-ray (blue arrow indicates the implanted support rod). Figure 18 Micro-CT 3D reconstruction panoramic image and slice image after taking samples from rabbit support rods (blue represents support rods; orange represents newly formed bone tissue); in the image, A represents the 6-week PTR group; B represents the 6-week PTRB group; C represents the 12-week PTR group; and D represents the 12-week PTRB group. Figure 19 The results of the Micro-CT statistical analysis for the PTR and PTRB groups are shown in the figure. (ns P > 0.05). In the figure, A represents the bone volume fraction at week 6 after implantation in the PTR and PTRB groups; B represents the trabecular thickness at week 6 after implantation in the PTR and PTRB groups; C represents the bone mineral density at week 6 after implantation in the PTR and PTRB groups; D represents the bone volume fraction at week 12 after implantation in the PTR and PTRB groups; E represents the trabecular thickness at week 12 after implantation in the PTR and PTRB groups; and F represents the bone mineral density at week 12 after implantation in the PTR and PTRB groups. Figure 20 Histological sections and VG staining results of two groups of rabbits implanted with support rods (black represents support rods, red represents collagen fibers): A is the 6-week PTR group; B is the 6-week PTRB group; C is the 12-week PTR group; D is the 12-week PTRB group. Detailed Implementation

[0014] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0015] Experimental materials used in this invention: Raw materials: Titanium alloy (Ti-6Al-4V) granular powder, specification: Ti64-53 / 20μm, provided by Weidu Xi'an Biotechnology Co., Ltd.; Mouse macrophages: raw264.7 cells, provided by Cybio Shanghai Biotechnology Co., Ltd. Experimental animals: 24 healthy male New Zealand rabbits weighing 2.5 kg to 3.5 kg, aged 6 to 8 weeks, were divided into 3 groups of 8 rabbits each, according to the design scheme. The rabbits were provided by the Department of Experimental Surgery, First Affiliated Hospital of Air Force Medical University, and the experiment has been approved by the Ethics Committee of Air Force Medical University.

[0016] The porous titanium alloy support rod of this invention was implanted in the femoral head after core decompression surgery to observe the osteogenic effect of the support rod with and without coating in the femoral head. The scaffold was implanted in the femoral condyle of a rabbit after drilling, to observe and compare the differences in osteogenic effect of three different internal pore structures in vivo. The discs were used only for cell experiments to observe the effects of coated and uncoated materials on in vitro cytotoxicity and proliferation. The discs and scaffolds with the same structure, porosity, average pore diameter, and average wire diameter as the porous titanium alloy support rod can be used to characterize the relevant properties of porous titanium alloy support rods with the same structure.

[0017] Example 1: A porous titanium alloy support rod and its application in rabbit femoral condyle implantation.

[0018] 1. Specification design of porous titanium alloy support rods, discs, and body structure brackets (hereinafter referred to as brackets). A porous titanium alloy support rod with a small beam was designed using CAD software. It includes a head and a tail that are fixedly connected. The head and body consist of a head and a body. Corresponding circular plates and body structure supports (hereinafter referred to as supports) with the same structure were designed for experimental research.

[0019] The specific parameters of the porous titanium alloy support rod are: (1) the porosity is maintained at 71.36±0.62%, the average pore diameter is 750.49±6.07μm, the average wire diameter is 371±15.3μm, the connectivity is 100%, and the error is determined to be 100μm based on the accuracy of the printer (BLT-S210). (2) The porous titanium alloy support rod is cylindrical in shape. Its head is a porous hemispherical structure with a diameter of 3.16 mm. The body is a mesh porous cylinder with a diameter of 3.16 mm and a length of 9.20 mm. The head and body are mesh porous beam structures. The mesh porous beam structure is composed of several hexagonal honeycomb structure units. The hexagonal honeycomb structure unit has a hexagonal structure around its perimeter and several hexagonal holes in the center. Several connecting beams are provided between the hexagonal honeycomb structure unit and the inner wall of the head and body. One end of the connecting beam is fixedly connected to the side of the hexagonal honeycomb structure unit, and the other end is fixedly connected to the inner wall of the head and body. (3) The tail is a solid cylinder with a diameter of 3.16 mm and a length of 2.50 mm. The perimeter is threaded with a thread pitch of 0.42 mm and a thread height of 0.25 mm. The bottom surface is an internal hexagonal locking hole.

[0020] The other parameters of the disc and the bracket are the same as those of the porous titanium alloy support rod. The disc and the bracket have a mesh-like porous beam structure inside: the bottom diameter of the disc is 12mm and the height is 4mm; the bottom diameter of the bracket is 3mm and the height is 4.5mm. The porosity and pore size are the same as those of the main structure of the porous titanium alloy support rod.

[0021] 3. Fabrication of porous titanium alloy support rods, discs, and body structure supports After the CAD software design process is completed, the model is saved in STL format. Selective Laser Melting (SLM) is used for printing, with the following parameters: upper epidermis laser power 90 W, scanning speed 1200 mm / s; infill laser power 350 W, scanning speed 900 mm / s; lower epidermis laser power 50 W, scanning speed 1300 mm / s. After printing, the model is cleaned with an ultrasonic cleaner, dried, and then sterilized with Co60 radiation for later use.

[0022] Comparative Example 1: A porous titanium alloy support rod and its preparation method.

[0023] The design is essentially the same as in Example 1, except that the porous titanium alloy support rod has the following specifications: porosity maintained at 74.53±0.30%, average pore diameter at 851.52±50.74 μm, and average wire diameter at 387±21.2 μm. The internal microstructure of the head and body of the porous titanium alloy support rod is a traditional rectangular structural unit. Similarly, a circular plate and a support with the same structural parameters as the porous titanium alloy support rod prepared in Comparative Example 1 were designed and fabricated.

[0024] Comparative Example 2: A porous titanium alloy support rod and its preparation method.

[0025] The design is essentially the same as in Example 1, except that the porous titanium alloy support rod has the following specifications: porosity maintained at 78.77±1.16%, average pore diameter at 804.96±32.20 μm, and average wire diameter at 317±23.1 μm. The head and body of the porous titanium alloy support rod have concentric trapezoidal structural units. Similarly, a circular sheet and support with the same structure as the porous titanium alloy support rod prepared in Comparative Example 2 were designed and fabricated.

[0026] Photographs were taken to record the structure and appearance of the porous titanium alloy support rods, discs, and brackets prepared in Example 1 and Comparative Examples 1-2, respectively. Figure 4 The porous titanium alloy support rods, discs, and body structure supports prepared in Example 1 and Comparative Examples 1-2 were characterized and tested. The specific methods and results are as follows:

[0027] I. Experimental Methods 1. Measurement of Pore Structure Data of Porous Titanium Alloy Support Rods: Four porous titanium alloy support rods prepared in Example 1 and Comparative Examples 1-2 were taken respectively and scanned using Micro-CT. The scanning parameters were set as follows: voltage 90kV, scanning rotation angle 360°, current 200μA, and scanning resolution 40μm. After scanning, three-dimensional reconstruction was performed using Nrecon and DataViewer software. A 10mm section of the porous structure of the rod body (excluding the hemispherical head and solid tail of the same diameter) was extracted. The extracted section was measured and analyzed using CTAn software to obtain the pore structure parameters, which were expressed in the form of x±s.

[0028] 2. In vitro mechanical property evaluation of porous titanium alloy support rods: A multi-functional static testing machine was used, and the machine parameters were set (compression speed: 1 mm / min). Based on the anatomy of the femoral head and the force distribution, and taking the average neck-shaft angle of 128° of the normal adult femur as the benchmark, the threaded portions of the porous titanium alloy support rods prepared in Example 1 and Comparative Examples 1-2 were respectively placed into a self-made fixture consisting of a wedge-shaped metal base and screws. Figure 5 (The instrument is fixed, and the testing machine is turned on to perform a three-point bending test to obtain the yield strength and stress-strain curve.)

[0029] 3. In vitro cell proliferation experiment: The porous titanium alloy discs prepared in Example 1 and Comparative Examples 1-2 were used as three groups, with 8 discs in each group. 5 x 10⁻⁶ discs were used. 4Cell suspensions of 100 μL / mL were seeded onto discs in well plates, with 2 mL of complete culture medium added to each well. The plates were then incubated. Three additional 24-well plates were divided into two groups (positive control and negative control), with eight wells in each group. No discs were placed in each group; 100 μL of cell suspension was seeded into each well, with 2 mL of complete culture medium added. The positive control group received 10 μL of complete culture medium containing 10% DMSO per well, and the negative control group received 10 μL of complete culture medium per well. The plates were removed on days 1, 4, and 7, and absorbance (A) was measured at 450 nm using the CCK-8 assay on an ELISA reader to observe the effects of the three groups on in vitro cell proliferation. Porous titanium alloy discs can characterize the properties of porous titanium alloy support rods with the same parameters.

[0030] 4. Rabbit femoral condyle implantation experiment (1) Surgical procedure: The implanted materials were sterilized by Co60 irradiation before the operation, and the instruments were sterilized by high temperature and high pressure steam. The rabbits were randomly divided into three groups, A, B and C, and the corresponding body structure scaffolds prepared in Comparative Example 1, Comparative Example 2 and Example 1 were implanted respectively, with 8 rabbits in each group.

[0031] The specific method is as follows: One randomly selected animal from the group is given a subcutaneous injection of Tylenol 100 at a dose of 0.1 mL / kg. After 2-3 minutes of anesthesia taking effect, the animal is weighed and its weight recorded. The animal is then shaved and its skin prepared, with the femoral condyle as the center, covering an area of ​​approximately 10 × 10 cm. 2 To maximize the exposure of the surgical area, the skin was routinely disinfected and draped, extending from the femoral condyle on the surgical side to the entire lower limb and foot, covering the prepared area. A longitudinal incision of approximately 1.5 cm was made on the lateral side of the femoral condyle using a scalpel. The attached fascia, tendons, and other soft tissues were then separated to fully expose the lateral femoral condyle. The fascia and periosteum on the surface of the condyle were carefully scraped away to expose the white bone. A hole was drilled perpendicular to the bone surface at the center of the lateral condyle using a bone drill, creating a cylindrical bone defect approximately 3 mm in diameter and 4.5 mm deep. A prepared porous titanium alloy scaffold was gently tapped into the bone defect until the entire scaffold was embedded in the bone. The surgical area was then irrigated with 3% hydrogen peroxide and 0.9% saline solution. The muscle tissue, fascia, and skin were sutured layer by layer, and the incision was disinfected with povidone-iodine. Serial labels were affixed to the rabbit ears for grouping. Post-surgery, administer cefazolin sodium injection 250mg intramuscularly once daily for 3 consecutive days. Once the rabbit regains consciousness, it can eat and move freely within its cage.

[0032] (2) General observation: After the operation, observe the general condition of each group of experimental rabbits, such as diet, defecation and mental state, and observe whether there are abnormal manifestations such as hematoma, exudation or infection in the wound.

[0033] (3) Gross observation of specimens: A total of 24 rabbits were used in three groups, with 8 rabbits in each group. They were sacrificed at weeks 6 and 12 to obtain femoral bones. The femoral condyles of the specimens were observed for any abnormalities such as effusion, abscess, or fracture. After collection, the specimens were cut 3 cm above the femoral condyle using a hard tissue slicer to ensure the integrity of the material and surrounding tissues. The specimens were placed in specimen bags and labeled. 50 mL of 4% paraformaldehyde fixative was poured into each specimen bag for fixation for 7 days.

[0034] (4) Micro-CT scan: Remove the specimen and place it on the Micro-CT scanning tray, securing it with masking tape to prevent movement. Place it in the instrument and close the chamber door. Set the scanning parameters as before and begin scanning. Use Nrecon and DataViewer software for 3D reconstruction, and CTAn software for measurement and analysis of the scan data. Select the condylar scaffold and its surrounding area as the region of interest, adjust the threshold and mark the color, and use CTVox software for 3D rendering and visualization analysis. Based on the obtained data and images, assess the ingrowth of bone tissue within the porous scaffold, the amount of new bone formation, and the integration of the scaffold with bone.

[0035] (5) Specimen fixation and embedding: Immerse the specimens in ethanol solutions of different concentrations (70%, 80%, 90%, 95%) once for each concentration, followed by two immersions in anhydrous ethanol and two immersions in xylene solution, with each immersion lasting 2 hours. Then, transfer the specimens sequentially to solutions I, II, and III (maintaining 4°C) and immerse them for 5 days each. Transfer the specimens to embedding bottles, label them, and slowly pour in methyl methacrylate, ensuring the liquid level completely submerges the specimen. Open the bottle cap and vacuum aspirate for 6 hours to remove air bubbles. Tighten the cap and place the bottle in a 50°C water bath for 48 hours, until the liquid inside the bottle solidifies into a transparent block of plastic.

[0036] (6) Histological sectioning and VG staining: The embedded block was removed and fixed on a hard tissue microtome. Sections were made horizontally along the cross-section of the scaffold, with a section thickness of approximately 300 μm. The sections were fixed to resin slides with glue, and excess glue was removed by squeezing. After 24 hours, the sections were ground. The sections were ground to a thickness of approximately 150 μm using a grinding machine, and the scratches were observed under a microscope. VG staining was performed and the sections were observed under a microscope.

[0037] 5. Statistical Analysis: Data obtained in this experiment were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and the SNK-q test was used for pairwise comparisons. All data were analyzed using SPSS 26.0 software, and a p-value < 0.05 was considered statistically significant.

[0038] II. Experimental Results 1. In vitro mechanical property evaluation of porous titanium alloy support rods The results of the three-point bending experiment are as follows: Figure 6 , Figure 7 As shown in Table 1, the maximum mechanical strength that the porous titanium alloy support rod prepared by this invention can bear is significantly better in both Comparative Example 2 and Example 1 than in Comparative Example 1, with statistically significant differences (P < 0.0001). However, the mean mechanical strength of Example 1 is higher than that of Comparative Example 2, with a statistically significant difference (P < 0.05). Comparative Example 2 has a higher standard deviation, indicating less stable mechanical properties. Therefore, the porous titanium alloy support rod prepared in Example 1 has the highest yield strength and the best mechanical properties, with an average load-bearing capacity of 713 N.

[0039] Table 1. Experimental data (N) of the maximum load-bearing capacity of the porous titanium alloy support rod prepared in this invention. Note: a indicates that the difference between this group and Comparative Example 1 is statistically significant, P<0.0001; b indicates that the difference between this group and Comparative Example 2 is statistically significant, P<0.05.

[0040] 3. In vitro cell proliferation experiment with porous titanium alloy support rods The results are as follows Figure 8 As shown, the absorbance of all three treatment groups and the negative control group increased with culture time on days 1, 4, and 7, indicating that the cell number in all four groups increased over time. The absorbance of the positive control group remained unchanged, indicating that the cell number did not change. At each time point, there were no statistically significant differences in absorbance values ​​between any two groups except the positive control group (P > 0.05).

[0041] 4. Experimental results of scaffold implantation in rabbit femoral condyle (1) General observation: One week after surgery, the rabbits in all three groups showed significant limitation of movement in the operated limbs and abnormal gait, which gradually improved. Two weeks after surgery, one rabbit in the comparative group died, and six weeks after surgery, material from one rabbit in Example 1 was found to have detached. The remaining animals did not show any obvious abnormalities after surgery until they were sacrificed for material collection.

[0042] (2) Gross observation of specimens: Except for one animal in three groups whose specimen material dislodged, no femoral condyle bone defects or fractures were found in the specimens of the other groups. One animal in the control group died in the second week. The surface of its gross specimen was covered with pale yellow soft tissue around the implantation area and the scaffold, with unclear boundaries from the surrounding tissues, which was considered to be purulent discharge caused by postoperative infection in the implantation area. Six weeks after the operation, one animal in the example group dislodged the scaffold material from the implantation area, which was located near the condyle and adhered to the tissue, which was considered to be dislodged during the animal's strenuous activity after the operation. The bone in the other specimens was uniform and continuous, with clear boundaries from the surrounding tissues, and bright and clear color. No infection, fracture, scaffold breakage, or dislodgement was found.

[0043] (3) Micro-CT scan: Data were compared based on three aspects: bone volume fraction, trabecular bone thickness, and bone mineral density in the scaffold implantation area. Results are as follows: Figure 8 As shown, at week 6, new bone tissue ingrowth was observed within the porous scaffold structures obtained in Example 1 and Comparative Examples 1-2, exhibiting differences in osteogenic formation. The differences in bone volume fraction, trabecular bone thickness, and bone mineral density were all statistically significant (P < 0.05). Specifically, the porous scaffold structure obtained in Example 1 showed significantly higher bone volume fraction, trabecular bone thickness, and bone mineral density after intervention compared to the other two groups (P < 0.05), indicating a significant bone ingrowth effect. Comparing Comparative Examples 1 and 2, although there was no statistically significant difference in trabecular bone thickness after implantation of the porous scaffold structure (P > 0.05), Comparative Example 1 still showed higher bone volume fraction and bone mineral density than Comparative Example 2, with statistically significant differences (P < 0.05). Overall, Comparative Example 1 showed the second best osteogenic effect, while Comparative Example 2 showed the worst. At week 12, bone ingrowth in all three regimens increased further compared to week 6, with increases in bone volume fraction, trabecular bone thickness, and bone mineral density. The bone volume fraction and trabecular bone thickness values ​​of Comparative Example 1 and Comparative Example 2 were nearly identical (P > 0.05), with statistically significant differences only in bone mineral density (P < 0.05). The bone volume fraction and bone mineral density values ​​of the porous scaffold obtained in Example 1 were still significantly higher than those of Comparative Example 1 and Comparative Example 2 (P < 0.05). While there was no statistically significant difference in trabecular bone thickness between Example 1 and Comparative Example 1 (P > 0.05), Example 1 was still significantly higher than Comparative Example 2 (P < 0.05). Comparison of bone mineral density values ​​showed that Example 1 was still significantly higher than Comparative Example 1-2 (P < 0.05), and Comparative Example 1 was also higher than Comparative Example 2 (P < 0.05).

[0044] (4) Histological sections and VG staining results: The results are as follows Figure 9As shown, at 6 weeks post-operation, all three groups of scaffold materials had red-stained collagen tissue and mineralized new bone tissue attached around them. In Comparative Example 1 and Example 1, a small amount of collagen fibers and yellowish-white new bone tissue were visible inside the pores of the scaffolds, while no obvious collagen fibers or new bone tissue were observed inside the pores of the scaffold in Comparative Example 2. Furthermore, in Comparative Example 1 and Comparative Example 2, there were still small gaps between the scaffolds and the surrounding tissue, and the internal tissue filling was relatively sparse, while no obvious gaps were observed between the scaffold and the new tissue in Example 1. At 12 weeks post-operation, obvious red collagen fibers and yellowish-white new bone tissue filled the pores of all three groups of scaffolds. In Comparative Example 1 and Example 1, the new collagen fibers and new bone tissue had filled more than half of the pore area inside the scaffolds, and some of the new collagen tissue had fused into cord-like structures and penetrated the scaffold pores. In Example 1, the new fibrous tissue and bone tissue not only penetrated the center of the scaffold material but also occupied most of the porous structure inside the scaffold, with large areas of new bone tissue fused together. In Comparative Example 1, fibrous and bone tissue had grown to the center of the scaffold material, but some areas within the pores remained without new tissue. In Comparative Example 2, while new collagen fibers and bone tissue also grew in, they were mainly concentrated at the edges of the pores, with relatively less ingrowth inside, occupying only a portion of the pores. The resulting new bone tissue was sparse and scattered. High-power microscopic observation of the interface bone integration revealed that in all three groups, the scaffold material, new collagen fibers, and bone tissue were tightly adhered to the periphery and inside the pores, with no obvious gaps. The histological analysis results showed a similar trend to the Micro-CT analysis results.

[0045] As can be seen from the above, the porous titanium alloy scaffolds prepared in Example 1 and Comparative Examples 1-2 of the present invention can effectively integrate with in vivo bone tissue and promote scaffold osteogenic formation in rabbit femoral condyle implantation experiments. Among them, the porous titanium alloy scaffold obtained in Example 1 showed significantly better osteogenic formation than that in Comparative Examples 1-2. The porous titanium alloy scaffold prepared in Example 1 had a bone volume fraction of 83.46% at 6 weeks and increased to 89.20% at 12 weeks, indicating that significant internal bone ingrowth effect was already shown in the early stage of scaffold implantation in Example 1.

[0046] Therefore, the porous titanium alloy implant material prepared by the hexagonal honeycomb structure unit porous titanium alloy support rod of Embodiment 1 of the present invention has better mechanical load-bearing performance and in vivo bone integration and bone ingrowth effect.

[0047] Example 2: Porous titanium alloy support rod (PTRB) with barium titanate coating and its functional characterization.

[0048] I. Experimental Materials and Methods 1. Raw materials: Titanium alloy (Ti-6Al-4V) particles (specification Ti64-53 / 20μm, Tektronix Plasma Systems Co., Ltd.); raw264.7 cells (mouse macrophages, Cybio Shanghai Biotechnology Co., Ltd.).

[0049] 2. Preparation of Barium Titanate Coated Porous Titanium Alloy Materials (1) The porous titanium alloy disc, bracket and porous titanium alloy support rod prepared in Example 1 are used as porous titanium alloy materials for later use.

[0050] (2) Preparation of barium titanate coating: Weigh 0.3714 g of ammonium fluoride and add it to 5 mL of deionized water. Add 200 mL of ethylene glycol and stir in a constant temperature magnetic stirrer to obtain a clear electrolyte. Use a platinum sheet as the cathode and porous titanium alloy material as the anode for anodic oxidation. The distance between the two electrodes is 20 mm, the voltage is 60 V, and the oxidation is carried out at room temperature for 30 minutes. Then, clean it in an ultrasonic cleaner and dry it. Place the oxidized porous titanium alloy material (circles, supports, and support rods) into a box-type resistance furnace and adjust the temperature to 400 °C at a heating or cooling rate of 1 °C / min. Hold it at the temperature for 3 hours for heat treatment. Prepare a 0.03 mol / L barium hydroxide solution with distilled water, pour it into an autoclave, and put the porous titanium alloy material in it to ensure that the solution is completely immersed in the porous titanium alloy material. Turn off the autoclave and heat it to 200 °C. After 2 hours, place it at room temperature to cool down. The polarization state of the porous titanium alloy material was changed using a piezoelectric polarization device with a polarization voltage of 11.5 kV and a polarization time of 30 minutes. After polarization, the material was cleaned with an ultrasonic cleaner, dried, and sterilized by Co60 radiation to obtain porous titanium alloy discs, porous titanium alloy supports, and porous titanium alloy support rods (PTRB) with barium titanate coatings, which were then ready for use.

[0051] 3. Characterization and testing of barium titanate coated porous titanium alloy materials (1) Micro-CT detection: Uncoated porous titanium alloy discs were used as the control group (PT), and barium titanate coated porous titanium alloy discs were used as the experimental group (PTB). Four discs were taken from each group and inserted vertically into foam plastic. They were then fixed to the Micro-CT scanning tray with masking tape, so that the cross-section of the disc was perpendicular to the long axis of the tray groove. The scanning chamber door was then closed. The scanning was performed according to the steps described above.

[0052] (2) SEM observation of surface morphology and EDS analysis: Two sets of porous discs were fixed to the SEM base with conductive adhesive and then transferred to the E-1010 gold spraying device for platinum spraying. The discs and base were placed in the field emission scanning electron microscope equipment and a vacuum was drawn. After completion, the surface morphology of the two sets of discs could be observed, the focus was adjusted and images were taken. EMAX ENERGY was used to analyze the elemental composition and distribution of the disc surface.

[0053] (3) Piezoelectric coefficient d 33 Measurement: Connect the measuring head and the instrument body, select the measurement range as x0.1, take out the plastic sheet from the accessory box and insert it between the upper and lower probes of the measuring head, adjust the handwheel at the top of the measuring head so that the plastic sheet is just pressed down, and select d on the instrument panel. 33 Preheat one side for 10 minutes, zero the circuit, remove the plastic sheet, and place two sets of porous discs between the upper and lower probes respectively. Adjust the handwheel to perform the test and obtain the piezoelectric coefficient (d). 33 ).

[0054] 4. Biological performance testing of barium titanate coated porous titanium alloy materials (1) Preparation of dynamic extract and CCK-8 cytotoxicity test: To simulate the effect of bone compression in vivo, a cyclic loading device was used to prepare dynamic extract of disc materials. One disc from each of the PT and PTB groups was placed in a 6-well plate, according to the national standard GB / T16886 (sample surface area / extract = 3 cm²). 2 / mL), add prepared complete culture medium (fetal bovine serum, DMEM high glucose medium, and penicillin-streptomycin mixture at a ratio of 10:90:1), and soak in an incubator at 37℃, 5% CO2, and saturated humidity for 72 hours. During this period, stimulate the discs with a circulating loading device for 1 hour daily for 3 consecutive days. The parameters of the circulating loading device were 1 Hz and 50 N (0.06 MPa). At 5 × 10⁻⁶ rpm... 4 Cell suspension at a density of 100 μL / mL was seeded into three 96-well plates, with 32 wells per plate. The plates were divided into four groups (PT group, PTB group, positive control group, and negative control group), with eight wells per group. Each well in the PT group received 10 µL of simple titanium alloy disc dynamic extraction solution, each well in the PTB group received 10 µL of barium titanate-coated titanium alloy disc dynamic extraction solution, each well in the positive control group received 10 µL of complete culture medium containing 10% dimethyl sulfoxide (DMSO), and each well in the negative control group received 10 µL of complete culture medium. After seeding, the plates were incubated. On days 1, 4, and 7, the plates were removed, and the absorbance (A) of each well was measured using the CCK-8 assay at a wavelength of 450 nm on an ELISA reader. The relative cell proliferation rate (%) is calculated using the formula: (Experimental group / Negative control group) × 100%. The toxicity level of the material is then determined based on this relative proliferation rate: 0% is Grade V, 1%-24% is Grade IV, 25%-49% is Grade III, 50%-74% is Grade II, 75%-99% is Grade I, and ≥100% is Grade 0. Grades 0 and I indicate no cytotoxicity, Grade II indicates mild cytotoxicity, Grades III and IV indicate moderate cytotoxicity, and Grade V indicates severe cytotoxicity.

[0055] (2) SEM observation of cell morphology: Take 3 discs each from the PT group and PTB group prepared above and place them in a 6-well plate. SEM is performed at 5×10⁻⁶ micrometers per ... 4 Two mL of cell suspension per well was seeded onto discs, and the discs were returned to the incubator for incubation for two days. During this period, the cell-disc complexes were stimulated using a circulating load device with the aforementioned parameters for one hour daily for two consecutive days. After incubation, 2.5% glutaraldehyde was added to each well and the discs were fixed at 4 °C for 24 hours. The cell-disc complexes were then removed, placed on a tray, and subjected to platinum spraying using an ion sputtering coating machine. Cell morphology was observed using SEM.

[0056] (3) Flow cytometry detection of apoptosis: Three discs from the PT group and three discs from the PTB group prepared above were placed in a 6-well plate and flowed at 5 × 10⁻⁶ cells / well. 4 Two mL of cell suspension per well was seeded onto each well of a disc, with three wells seeded as a blank control. After seeding, the discs were incubated together in a cell culture incubator for 48 hours. During this period, both disc groups were stimulated for 1 hour daily using a circulating loading device for two consecutive days. After two days, the discs were removed, the culture medium was aspirated, and the cells were resuspended after trypsin digestion. The cells were centrifuged, the supernatant was discarded, and 2 mL of PBS was added. The cells were washed at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL (1x) binding buffer. 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added, mixed, and incubated at 4°C in the dark for 30 minutes. The cells were then analyzed by flow cytometry.

[0057] (4) Detection of cell proliferation activity by Calcein-AM / PI staining: Take 3 discs each from the PT group and PTB group prepared above and place them in a 6-well plate. Stain with 5×10⁻⁶ ppm. 4 Two mL of cell suspension per well was seeded onto discs and incubated for 3 days, with stimulation for 1 hour daily using a circulating loading device (parameters as before). After incubation, the discs were removed, the culture medium was aspirated, and the cells were resuspended after trypsin digestion. The discs were centrifuged, the supernatant was discarded, and cell viability was measured using a Calcein-AM / PI kit. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were simultaneously detected under a fluorescence microscope using a 490 nm excitation filter. Two fields of view were randomly selected from each well for photographing, and fluorescence intensity was analyzed using ImageJ.

[0058] (5) TRAP staining to detect osteoclast-inducing activity of cells: Take 3 discs each from the PT group and PTB group prepared above and place them in a 6-well plate. Stain with 5×10⁻⁶ T cells. 42 mL of cell suspension per well was seeded onto discs and incubated for 2 days. During this period, the cells were stimulated for 1 hour daily using a circulating loading device (parameters as before) for 2 consecutive days. After the incubation, the discs were removed, 2 mL of trypsin was added to each well, and the discs and discs were thoroughly digested by pipetting for 1 minute. The discs were centrifuged, and the cells were resuspended in complete culture medium and seeded into a new 6-well plate. The plates were then incubated in a cell culture incubator for 12 hours. Subsequently, 500 μL of complete culture medium containing RANKL (50 ng / mL) was added to each well for induction. The medium was changed every 24 hours. After 5 days of induction culture, the cells were stained using a TRAP staining kit. The cells were observed and counted under a microscope. The counting criteria were: (1) 8 fields of view were randomly selected for counting under 10x magnification without repetition; (2) The cytoplasm of mature osteoclasts was rose red or pink after TRAP staining, and the number of cell nuclei was ≥3.

[0059] 5. Statistical analysis: Quantitative data are expressed as mean ± standard deviation (...). ±s) indicates the mean. One-way ANOVA was used for comparisons between groups, the SNK-q test was used for pairwise comparisons between groups, and the chi-square test was used for comparisons of rates. 2 Statistical analysis was performed on the data using SPSS 26.0 software. P <0.05 indicates a statistically significant difference.

[0060] II. Experimental Results 1. Surface characterization and analysis results of barium titanate coated porous titanium alloy materials (1) Micro-CT scan results: The results are shown in Table 2. After the barium titanate coating, the pore size and porosity of the structure decreased slightly, while the wire diameter increased slightly. There was no statistically significant difference between the two sets of data (P<0.05). The uniformly coated barium titanate particles had little effect on the porous and beam structure of the titanium alloy.

[0061] Table 2 Pore structure parameters of micro-CT discs in PT and PTB groups (2) SEM observation and EDS analysis: The results are as follows Figure 10 As shown, barium titanate particles are uniformly coated on the surface and inside the voids of the disc, bonding tightly with the titanium alloy and exhibiting a granular and plate-like crystalline structure, forming a certain degree of surface roughness, while the surface of pure titanium alloy is smoother and more even. Figure 11 As shown, the area where the signals of elements Ti, Al, and V overlap represents the main body of the disc, while element Ba represents the surface coating.

[0062] (3) Detection of piezoelectric coefficient d33: such as Figure 12 Quasi-static d 33 The measuring instrument measures the piezoelectric coefficient d of the PTB disc. 33The value was 0.688 ± 0.042 pC / N, while it was not detected in the PT group discs.

[0063] 2. Results of biological performance testing (1) Dynamic extraction solution detection of cell proliferation and toxicity: The results are shown in Table 3. The absorbance values ​​of the PT group, PTB group and negative control group all increased over time, and the relative proliferation rate remained above 90%, indicating good cell proliferation activity and an increase in cell number over time. However, the absorbance value of the positive control group showed a decreasing trend over time, indicating that the cells had basically lost their proliferative activity and the cell number continued to decrease. Pairwise comparisons of the absorbance values ​​of the PT group, PTB group and negative control group showed no statistically significant differences (P>0.05), which can be considered that the extracts of the PT group and PTB group prepared by this process have no effect on in vitro cell proliferation.

[0064] Table 3. Absorbance values ​​(x̅±s) and cytotoxicity levels at different time points during cell co-culture with dynamic extract (n=8) (2) SEM observation of cell state on material surface: such as Figure 13 As shown, the cells exhibited good growth on both groups of metal surfaces, with plump cell morphology, continuous and intact cell membranes, and good adhesion to the materials. In the PT group, the surface cells were spherical or ellipsoidal, extending pseudopodia to grow and adhere to the titanium alloy metal particle surface, with most cells adhering to the gaps between the surface metal particles. The pseudopodia were numerous and relatively slender. In contrast, the surface cells in the PTB group showed an approximately flattened or spindle-shaped extended or semi-extended state, with slightly fewer pseudopodia and generally adhering to the material surface. The cell density was slightly higher, and some areas were observed to be covered by a large number of cells forming a cell layer. The barium titanate nanoparticles created a certain degree of roughness on the titanium alloy surface, providing attachment points for cell adhesion and growth.

[0065] (3) Apoptosis rate detection: such as Figure 14 As shown, region B1 represents cells that have died due to other reasons or cell fragments without cell membranes; Annexin V-FITC cannot label these cells, but PI can. Region B2 represents late-stage apoptotic cells; both Annexin V-FITC and PI can label these cells. Region B3 represents normal living cells; neither Annexin V-FITC nor PI can label these cells. Region B4 represents early-stage apoptotic cells; Annexin V-FITC can label these cells, but PI cannot. The apoptosis rate is the sum of (B2 + B4). Data analysis showed that the apoptosis rates of the PTB group and the PT group were similar and slightly higher than those of the blank control group, but the difference was not statistically significant compared with the blank control group (P > 0.05). It can be considered that the two groups of materials had no effect on the apoptosis rate after in vitro co-culture with loading.

[0066] (4) Observation of cell viability by Calcein-AM / PI staining: The staining results are as follows Figure 15 As shown, Figure 15 AC and Figure 15 The images shown in the images (DF) are Calcein-AM staining, PI staining, and fusion images of the PT and PTB groups, respectively. Both groups showed green fluorescence (representing live cells) and red fluorescence (representing dead cells), with green fluorescence exceeding red fluorescence in both groups. Image J analysis of fluorescence intensity revealed that the fluorescence intensity of Calcein-AM staining, PI staining, and the fusion image in the PTB group was significantly higher than that in the PT group, with statistically significant differences (P < 0.05). This indicates that after co-culture with the loading, the PTB group showed a significantly increased cell number and stronger cell proliferation activity, while also exhibiting a slightly higher number of apoptotic cells.

[0067] (5) Osteoclast induction and TRAP staining: After co-culture with cell load, osteoclast induction and TRAP staining were performed, and cells were counted under a microscope to understand the effect of the material on macrophage osteoclast differentiation. The results are as follows: Figure 16 As shown in the microscopic images, both groups exhibited a large number of mononuclear / macrophages with purplish-red nuclei. Most were round or nearly round, with a few exhibiting varying morphologies, and pseudopodia were visible around the nuclei. Large osteoclasts were also observed, containing varying numbers of bluish-purple nuclei, with pale red or light purple cytoplasm. Cell counting and statistical analysis based on the microscopic images revealed a significantly higher total cell count in the PTB group compared to the PT group (P < 0.0001), a result corroborated by Calcein-AM / PI staining. The osteoclast count was significantly lower in the PTB group than in the PT group (P < 0.01). Furthermore, the percentage of osteoclasts in the total cell count was significantly lower in the PTB group than in the BT group (P < 0.05). This indicates that co-culturing the PTB material with cell load in vitro effectively promoted cell proliferation while simultaneously reducing the osteoclast differentiation capacity of macrophages.

[0068] In summary, the barium titanate piezoelectric ceramic-coated porous titanium alloy material not only compensates for the insufficient bone ingrowth defect of porous titanium alloys in vivo, but also generates a local micro-electric field effect similar to that of natural bone tissue to achieve a biomimetic effect.

[0069] Example 3: Evaluation of the effect of barium titanate coated porous titanium alloy support rod in repairing femoral head necrosis in rabbits.

[0070] I. Experimental Materials and Methods 1. Animals and grouping: Twenty healthy adult male New Zealand rabbits weighing approximately 3 kg and aged 6-8 weeks were selected. The experimental rabbits were provided by the Department of Laboratory Surgery, First Affiliated Hospital of Air Force Medical University. The experiment has been approved by the Ethics Committee of Air Force Medical University.

[0071] 2. Establishment of a rabbit femoral head necrosis animal model: Twenty male New Zealand rabbits were used for the experiment, housed individually in cages with free access to food and water in a well-ventilated environment, and fed a routine acclimatization diet for one week. First, LPS (10 μg / kg) was injected intravenously via the ear vein. Twenty-four hours later, MPS (20 mg / kg / day) was injected into the gluteal muscle near the femoral head, for a total of three days. One day after the MPS injection, cefazolin sodium (250 mg / day) and pantoprazole injection (40 mg / day) were administered via gluteal injection for seven consecutive days. After the injections, the mental state, gait, food intake, urination and defecation, and fur of each group of animals were observed daily. The time and number of deaths were recorded, and the animals were dissected and the cause of death was analyzed.

[0072] 3. Decompression of rabbit femoral head core and implantation of support rod (1) Surgical procedure: Sixteen early ONFH model rabbits were randomly divided into two groups. The uncoated porous titanium alloy support rod (PTR) prepared in Example 1 and the barium titanate coated porous titanium alloy support rod (PTRB) prepared in Example 2 were implanted in the two groups, with 8 rabbits implanted in each group. One animal in each group was randomly selected and subcutaneously injected with Tylenol 100 at a dose of 0.1 mL / kg. After 3 minutes of anesthesia, the animal was weighed and the weight was recorded. The skin was shaved with the posterior edge of the greater trochanter of the femur as the center, covering an area of ​​approximately 15 × 15 cm. 2 Routine disinfection draping; disinfection area 13×13cm centered on the greater trochanter of the femur. 2The surgical area extends from the spine on the surgical side superiorly to the entire foot inferiorly, from the lower abdomen anteriorly to the tail posteriorly. A longitudinal incision, approximately 2.5 cm long, is made in the skin at the posterior border of the greater trochanter of the femur. The superimposed muscles are dissected close to the posterior border of the greater trochanter, fully exposing the area from the posterior side of the greater trochanter to the femoral neck. The fascia and periosteum on the posterior surface of the greater trochanter are carefully dissected to expose the femoral neck. A simple positioning point is established at the midpoint of the line connecting the greater and lesser trochanters, slightly off-center from the femoral neck. A 0.8 mm drill bit is used to drill into the femoral head, maintaining an angle of approximately 20° between the drill bit and the femoral surface. The drilling depth is approximately 12 mm. After drilling, the drill bit is reversed and removed, and a 0.8 mm positioning pin is inserted. The surgical area is covered with sterile gauze. Anterior and lateral fluoroscopy is performed at the bedside to observe the drilling depth and direction, adjusting as needed until the drill penetrates approximately 1-2 mm below the femoral head cartilage. The hole was enlarged using 1.5mm and 3.0mm hollow drill bits, with the drilling depth adjusted to 10mm, creating a cylindrical core decompression channel approximately 3mm in diameter and 10mm deep. The two sets of porous titanium alloy support rods were then hammered into the decompression channel. When the tail entered the cortical bone, it could be tightened using the internal hexagonal holes. After completion, the surgical area was rinsed with 3% hydrogen peroxide and 0.9% saline. The muscles, fascia, and skin were sutured sequentially, and the incision was disinfected. A serial number label was affixed to the rabbit's ear for group identification. Postoperatively, cefazolin sodium injection 250mg / time was administered intramuscularly once daily for 3 consecutive days. The rabbit could eat and move freely within its cage after regaining consciousness. Postoperative X-ray of rabbit femoral head core decompression and support rod implantation is shown below. Figure 17 As shown. (2) General observation: After the operation, observe the general condition of each group of animals, including diet, defecation, gait and mental state, and observe whether there is any abnormal manifestation such as suppuration, infection and exudation in the wound. (3) Gross observation of specimens: There are 16 experimental rabbits in two groups, 8 in each group. They were sacrificed at the 6th and 12th weeks to obtain femoral tissue. Observe whether there is any abnormal condition such as effusion, abscess or fracture in the greater trochanter of the femur. After the femoral specimens were obtained, they were placed in specimen bags and labeled. 50 mL of 4% paraformaldehyde fixative was poured into each specimen bag to fix the condylar specimen for 7 days. (4) Micro-CT detection: According to the steps described in the above example, micro-CT was used to evaluate and analyze the ingrowth of bone tissue and new bone in the porous support rod. (5) Specimen fixation and embedding: The experimental steps are the same as in Example 1. (6) Histological sectioning and VG staining: The specific steps are the same as in Example 1.

[0073] 4. Statistical Analysis: The data obtained in this experiment, including continuous data, are expressed as mean ± standard deviation (SD). Data are expressed as mean ± standard deviation (s), and one-way ANOVA was used for comparisons between groups. All data were analyzed using SPSS 26.0 software. P <0.05 indicates a statistically significant difference.

[0074] II. Experimental Results 1. Results of establishing a rabbit femoral head necrosis model: From day 3 of modeling, experimental rabbits generally exhibited hair loss, decreased food intake, and reduced activity. During week 1, fur was disheveled, food intake increased slightly, but activity remained low; 5 rabbits developed diarrhea, and 1 died on day 9, presumably due to diarrhea or infection. In week 2, rabbits generally experienced severe hair loss, reduced subcutaneous fat and muscle tissue, and unchanged food intake; 6 rabbits developed diarrhea, with 1 dying on day 15 and 16 respectively, presumably due to the same cause of death. By week 5, some rabbits exhibited abnormal gait and lameness.

[0075] 2. Biological test results after core decompression and support rod implantation in rabbit femoral heads (1) General observation: 1-2 weeks after surgery, both groups of animals showed significant limitation of movement in the operated limbs, abnormal gait, and lameness, which later improved. One animal in group A died 3 weeks after surgery. The remaining animals did not show any obvious abnormalities after surgery until they were euthanized for tissue collection.

[0076] (2) Gross observation of specimens: No femoral condyle bone defects or fractures were found in any of the specimens. The bone in each specimen was uniform and continuous, with clear boundaries from the surrounding tissues, and bright and clear color. No infection, fractures, or support breakage or dislodgement were found. In the PTR group and PTRB group, one femoral head support rod was implanted slightly lower, with the head of the rod located about 4-5 mm below the femoral head. The rest were normal.

[0077] (3) Micro-CT scan: Results are as follows Figure 18 and Figure 19As shown, at week 6, the bone volume fraction in both groups was close to 80%, the trabecular bone thickness was close to 0.9 mm, and the bone mineral density was approximately 0.7 g / cm³, indicating that there was significant ingrowth of new bone tissue into the support rods in both groups during the early implantation period. Secondly, while the mean values ​​of the three data points were slightly higher in the PTR group than in the PTRB group, the difference was not statistically significant (P < 0.05). At week 12, the bone volume fraction, trabecular bone thickness, and bone mineral density in the PTRB group gradually increased, with the mean bone volume fraction approaching 100%, indicating that the new bone tissue had essentially filled the pores of the rod at this time point, a statistically significant difference compared to the PTR group at the same time point (P < 0.05). Furthermore, the bone mineral density in both groups also increased compared to week 6, but the increase was more significant in the PTRB group, showing a statistically significant difference compared to the PTR group (P < 0.01). While both groups showed an increase in trabecular bone thickness, no statistically significant difference was observed (P < 0.05). As can be seen from the cross-sectional images of the 3D reconstruction rendering, at week 6, new bone ingrowth was observed in the pores of the rod in both groups. More bone growth was observed near the edges, while some pores in the center remained unfilled. The amount of bone in the pores of the PTRB group was slightly higher than that of the PTR group. At week 12, both groups had newly formed trabeculae filling the pores around and inside the rods, with most pores tightly filled. However, in the PTR group, some pores near the head of the support rod remained unfilled, while the PTRB group showed satisfactory filling of the pores with new bone tissue.

[0078] (4) Histological sections and VG staining: such as Figure 20 As shown, at 6 weeks post-surgery, both groups of support rods showed red-stained collagen tissue and mineralized new bone tissue attached to their periphery. A small amount of collagen fibers and new bone tissue were visible inside the pores near the edges. High-power microscopy revealed that the interface between the new tissue and the material was tightly integrated with the bone in both groups, but the internal tissue filling was relatively sparse, with no visible difference to the naked eye. At 12 weeks post-surgery, both groups showed obvious red collagen fibers and white new bone tissue filling the pores, with the new bone tissue filling nearly half of the internal pores. From the head to the tail of the support rod, the new collagen tissue had fused into cord-like structures and penetrated the internal pores of the rod, with the central area of ​​the support rod also occupied by new tissue. However, in the PTR group, although new fibrous tissue and bone tissue were visible in the central area, a significant portion of the pores remained unoccupied. Bone integration was similar to that at 6 weeks; in both groups, the support rods, new collagen fibers, and bone tissue were tightly adhered to their periphery and inside the pores, with no obvious gaps. The histological analysis results showed the same trend as the Micro-CT analysis results. Therefore, the barium titanate piezoelectric ceramic porous titanium alloy support rod prepared by this invention can generate an effective force-electric conversion effect in vivo, thereby promoting osteoogenesis. This support rod material is expected to be used to solve problems such as decreased femoral head mechanical properties and difficulty in osteogenic repair after ONFH core decompression surgery.

[0079] It should be noted that the dimensions of the porous titanium alloy support rod in Embodiment 1 of this invention are those used for rabbit femoral condyle implantation experiments and core decompression surgery. For different animals, the dimensions can be adjusted as needed, maintaining the porosity, average pore diameter, average wire diameter, and basic structure unchanged, while the length and diameter can be adaptively adjusted. Therefore, the size parameters set in this invention should not be considered as limitations of the invention. The diameter of the porous titanium alloy support rod is determined based on the femoral neck diameter of each animal, and the length is determined based on the insertion position of the support rod.

[0080] The dimensional parameters of porous titanium alloy support rods used in sheep and humans can be set as follows: Sheep body: Total length 47±4mm, body length 37±4mm, tail length 10mm, diameter 6±1mm. Tail length 10mm, thread pitch 1mm, thread height 0.5mm.

[0081] Human body: Total length 100mm±10mm, head and body length 85±10mm, tail 15±5mm, diameter 10±2mm, thread spacing 2mm, thread height 1mm.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0083] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A porous titanium alloy support rod, characterized in that, The porous titanium alloy support rod includes a head and a tail that are fixedly connected. The head is a porous cylindrical mesh, comprising a head and a body. The head and body are porous mesh beam structures composed of several hexagonal honeycomb structural units. The tail is a solid cylinder. Each hexagonal honeycomb structural unit has a hexagonal periphery and several hexagonal holes in its center. Several connecting beams are provided between the hexagonal honeycomb structural unit and the inner wall of the head. One end of each connecting beam is fixedly connected to the side of the hexagonal honeycomb structural unit, and the other end is fixedly connected to the inner wall of the head. The porous titanium alloy support rod has an average pore diameter of 700μm to 800μm, a porosity of 70% to 75%, and an average wire diameter of 340μm to 400μm.

2. The porous titanium alloy support rod according to claim 1, characterized in that, The porous titanium alloy support rod has an average pore diameter of 740μm to 760μm and a porosity of 70% to 73%. The tail has a diameter of 3mm to 14mm and a length of 2mm to 10mm. A hexagonal locking hole is provided on the bottom surface of the tail. Threads are provided around the periphery of the tail. The thread pitch is 0.4mm to 2.2mm and the thread height is 0.2mm to 1.2mm.

3. The porous titanium alloy support rod according to claim 1, characterized in that, The head is a porous hemispherical structure with a diameter of 3mm to 12mm, and the body is a mesh porous cylinder with a diameter of 3mm to 12mm and a length of 9mm to 110mm.

4. A method for preparing a porous titanium alloy support rod according to any one of claims 1-3, characterized in that, The CAD software is used to design the porous titanium alloy support rod structure, and then the laser selective melting forming technology is used for printing, and the printing parameters are as follows: the upper skin laser power is 80W-100W, the scanning speed is 1000mm / s-1300mm / s, the inner filling laser power is 300W-400W, the scanning speed is 800mm / s-1000mm / s, the lower skin laser power is 40W-60W, the scanning speed is 1200mm / s-1400mm / s, after printing, the ultrasonic cleaning machine is used for cleaning, and after drying 60 Co radiation sterilization standby.

5. The method for preparing the porous titanium alloy support rod according to claim 4, characterized in that, The printing parameters are as follows: upper epidermis laser power 90 W, scanning speed 1200 mm / s; inner filling laser power 350 W, scanning speed 900 mm / s; lower epidermis laser power 50 W, scanning speed 1300 mm / s.

6. A porous titanium alloy support rod with a barium titanate coating, characterized in that, The barium titanate coated porous titanium alloy support rod is obtained by coating the surface of the porous titanium alloy support rod according to any one of claims 1-3 with a barium titanate coating.

7. A method for preparing a barium titanate coated porous titanium alloy support rod according to claim 6, characterized in that, A clear electrolyte was obtained by mixing ammonium fluoride with deionized water and ethylene glycol at a ratio of 0.3714 g to 0.4 g: 5 mL: 200 mL. Using a platinum sheet as the cathode and a porous titanium alloy support rod as the anode, anodizing was performed. After cleaning and drying, the temperature was increased to 400℃ at a rate of 0.5℃ / min to 1.5℃ / min and held for 2.5h to 3.5h, followed by heat treatment. Then, the heat-treated porous titanium alloy support rod was immersed in barium hydroxide solution and heated to 200℃ to 220℃, held for 1.5h to 2.5h, and then cooled to room temperature at a rate of 0.5℃ / min to 1.5℃ / min. Finally, it was polarized at a voltage of 10kV to 13kV for 30min. After polarization, it was cleaned and dried. 60 Co-irradiation sterilization yielded a barium titanate-coated porous titanium alloy support rod.

8. The method for preparing a barium titanate coated porous titanium alloy support rod according to claim 7, characterized in that, After anodizing, cleaning, and drying, the temperature was increased to 400℃ at a rate of 1℃ / min and held for 3 hours for heat treatment. Then, the heat-treated porous titanium alloy support rod was immersed in a 0.03mol / L barium hydroxide solution, heated to 200℃, held for 2 hours, and then cooled to room temperature at a rate of 1℃ / min. Finally, it was polarized at a voltage of 11.5kV for 30 minutes to complete the polarization.

9. The use of a porous titanium alloy support rod according to any one of claims 1-3 or a porous titanium alloy support rod with a barium titanate coating according to claim 6 in the preparation of bone graft materials.

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