Preparation method of surgical titanium alloy fusion cage active coating

Abstract

The invention discloses a preparation method of an active coating of a surgical titanium alloy fusion cage. The preparation method comprises the following steps: S1, preparing a titanium alloy fusion cage with a porous structure through 3D printing; s2, constructing a polydopamine coating on the surface of the titanium alloy fusion cage; s3, preparing a calcium phosphorus deposition solution, and performing hydroxyapatite mineralization on the polydopamine coating; and S4, grafting TBMP2 protein on the surface of the hydroxyapatite mineralized coating to complete the preparation of the titanium alloy fusion cage active coating. The problems that an existing titanium alloy fusion cage is poor in biological inertness and osseointegration performance and has many side effects in BMP2 application are solved, and the titanium alloy fusion cage can adapt to personalized implantation requirements.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, and particularly relates to a preparation method of an active coating of a surgical titanium alloy fusion device. BACKGROUND

[0002] Scoliosis, degenerative disc disease and other spine-related diseases seriously endanger the health of patients, and full and good fusion is the key to the treatment of the diseases. The appearance of intervertebral fusion devices greatly promotes the development of intervertebral fusion technology. The intervertebral fusion device can avoid complications such as bone block shedding caused by the use of autologous bone grafting alone, can produce intervertebral distraction-compression effect, maintain the stability of the surgical segment, and provide a good environment for intervertebral fusion.

[0003] With the continuous development of material science, titanium alloy fusion devices have been widely used in intervertebral fusion. However, due to the differences in body size, anatomical structure and other differences of patients, the standard preparation of the fusion device often cannot meet the clinical needs. From a higher technical requirement, the best treatment method should be personalized treatment, and the best implant should be a personalized implant. The emergence of 3D printing technology provides reliable technical support for the manufacture and wide application of personalized implants. However, the surface of titanium alloy is very smooth, which is not conducive to the adhesion of cells and tissues. And titanium alloy belongs to inert metal, which cannot induce osteointegration with the surrounding bone tissue. Long-term implantation may cause loosening, pseudo joint formation, wear, aseptic inflammation and other problems. Therefore, constructing a stable active coating on the surface of titanium alloy is the key to the application of titanium alloy devices.

[0004] At present, many methods have been developed for the surface modification of titanium alloy, such as thermal spraying, plasma treatment, acid-base treatment, electrodeposition, laser surface modification, physical vapor deposition, micro-arc oxidation, and biomimetic mineralization treatment. Among them, biomimetic mineralization treatment refers to depositing a calcium and phosphorus mineralized coating on the surface of titanium alloy, which can play the excellent bone induction ability of calcium phosphate itself, and through ion exchange with the body fluid around the implant, Ca 2+ and PO4 3- are dissolved out, the ions in the environment are re-deposited, and bidirectional bone formation is achieved. Hydroxyapatite (HA) has a chemical composition similar to bone mineral and is the most widely used calcium phosphate salt, which has the ability to promote osteogenic differentiation, inhibit inflammatory response, and enhance the response of initial bone tissue. However, how to prepare a uniform and dense coating on the pore surface of a smooth and porous scaffold is still a challenge. Molecular self-assembly enables polymer molecules to be uniformly anchored to the inner and outer surfaces of the porous structure and form a self-assembled molecular layer combined with the substrate. Polydopamine (PDA) is a typical self-assembled coating polymer that can adhere to the surface of most biomaterials regardless of their size and shape. In addition, the PDA coating can act as a bridge to further react with other compounds to form a composite coating.

[0005] In addition, to enhance the bone inductive properties of the titanium alloy fusion instrument, the addition of BMP2 factor is an effective method. BMP2 is the first powerful osteogenic factor approved by the US FDA, and its use with other materials can effectively promote bone formation. However, the high osteogenic activity of BMP2 depends on the maintenance of an effective concentration of BMP2 locally in the bone graft. However, conventional BMP2 often dissolves rapidly at the initial stage of implantation, resulting in explosive release, rapid diffusion into the surrounding tissue and the whole body, and a series of side effects, including vertebral body osteolysis, heterotopic ossification, acute inflammation, etc.

[0006] Therefore, how to construct a bioactive coating on the surface of the titanium alloy fusion instrument, which has good bone conduction and bone induction properties, while avoiding the side effects of BMP2 application, is a key problem to improve the clinical application effect of 3D printed titanium alloy fusion instrument. SUMMARY

[0007] Based on the problems in the background art, the present application provides a preparation method of a surgical titanium alloy fusion instrument active coating, which solves the problems of poor bioinertness and bone integration of existing titanium alloy fusion instruments, and many side effects in the application of BMP2, and can adapt to the demand of personalized implantation.

[0008] The present application is implemented by the following technical solutions: A preparation method of a surgical titanium alloy fusion instrument active coating, comprising the following steps: S1. Preparing a porous titanium alloy fusion instrument by 3D printing; S2. Constructing a polydopamine coating on the surface of the titanium alloy fusion instrument; S3. Preparing a calcium and phosphorus deposition solution and performing hydroxyapatite mineralization on the polydopamine coating; S4. Grafting TBMP2 protein on the surface of the hydroxyapatite mineralized coating, thereby completing the preparation of the titanium alloy fusion instrument active coating.

[0009] Preferably, in step S1, the method for 3D printing the titanium alloy fusion instrument is: relying on CT or MRI medical image data to reconstruct a three-dimensional model of the vertebrae to be fused; inputting the data into a 3D printing system and using selective laser melting technology to prepare a porous titanium alloy fusion instrument.

[0010] Preferably, the process of constructing the polydopamine coating in step S2 is: dissolving polydopamine in a 30% ethanol / ultra-pure water solution to prepare a polydopamine coating deposition solution, placing the titanium alloy fusion instrument in the polydopamine coating deposition solution, and incubating at 37°C for 24 hours; replacing the deposition solution and incubating again at 37°C for 24 hours; ultrasonic cleaning to remove unbound polydopamine.

[0011] Preferably, the concentration of the polydopamine coating deposition solution is 1-4 mg / ml.

[0012] Preferably, the calcium-phosphorus deposition solution in step S3 specifically comprises 6.8-7.5 mM Ca(NO3)2·4H2O, 3.8-4.5 mM NaH2PO4·2H2O and 1.6-2.4 mM NaHCO3.

[0013] Preferably, the process of performing hydroxyapatite mineralization on the polydopamine coating in step S3 is specifically as follows: placing the fusion device with the polydopamine coating on the surface obtained in step S2 in a calcium-phosphate deposition solution, and incubating at 37°C for 12 hours; replacing the calcium-phosphate deposition solution, and incubating again at 37°C for 12 hours; and drying at 37°C.

[0014] Preferably, the process of grafting TBMP2 protein in step S4 is specifically as follows: preparing a TBMP2 solution with a concentration of 0.1-1 mg / ml; placing the mineralized fusion device obtained in step S3 in the TBMP2 solution and incubating it at 4°C for 24 hours; taking it out and freeze-drying it for storage.

[0015] Preferably, the TBMP2 is a targeted bone morphogenetic protein 2 expressing a (DSS)6 sequence at the N-terminus of the native BMP2 sequence.

[0016] Preferably, the porous titanium alloy fusion device has a porosity of 30%-70% and a pore size of 200-600 μm.

[0017] The present invention also discloses a 3D printed bioactive titanium alloy fusion device prepared by the preparation method, and discloses the use of the 3D printed bioactive titanium alloy fusion device in the preparation of a spinal fusion implant, wherein the spinal fusion implant is used for cervical or lumbar intervertebral fusion.

[0018] Beneficial effects of the present invention: 1. This invention solves the technical problem of constructing a uniform and stable mineralized coating on the surface of a porous titanium alloy fusion device by using a PDA-mediated HA mineralization method; 2. The present invention utilizes the (DSS)6 sequence expressed at the N-terminus of BMP2 to enable it to acquire the ability to specifically bind to HA, enabling it to combine with the HA mineralized coating to achieve a sustained release effect, thus avoiding the problem of explosive release of conventional BMP2; 3. In the active coating constructed by the present invention, the PDA layer, HA mineralized layer, and TBMP2 form a synergistic mechanism of action: PDA provides the ability to tightly bond with the titanium alloy surface, while the HA mineralized coating and TBMP2 exert osteoconduction and osteoinduction effects, enhancing bone integration at the fusion device-bone interface, thereby achieving long-term and stable implant results; 4. The 3D-printed titanium alloy fusion cage with an active coating prepared by the present invention can not only meet the needs of personalized implantation, but also effectively promote bone integration at the cage-bone interface, thereby improving the success rate of intervertebral fusion; 5. After release, the TBMP2 used in the present invention can act on the vertebral bone tissue rich in HA, reducing the risk of spreading to other tissues, effectively alleviating the side effects of vertebral osteolysis, ectopic osteogenesis, acute inflammation, etc. caused by conventional BMP2, and is conducive to the long-term implantation of the fusion device. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are used to further explain the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 Schematic diagram of the preparation method of the active coating of the surgical titanium alloy fusion device of the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described in detail below with reference to specific embodiments, but the protection scope of the present invention is not limited to the following embodiments. Example 1

[0021] Preparation of PDA-mediated HA mineralization coating S1. Fabrication of porous titanium alloy fusion cages by 3D printing. A Ti6Al4V titanium alloy porous fusion cage was prepared by 3D printing using selective laser melting technology, with a porosity of 50% and a pore size of 400 μm.

[0022] S2. Constructing a polydopamine coating on the surface of a titanium alloy fusion cage; Dopamine hydrochloride (PDA) was dissolved in 30% ethanol / ultrapure water solution to prepare a PDA coating deposition solution with a concentration of 2 mg / ml. A titanium alloy fusion device was placed in the PDA coating deposition solution and incubated at 37°C for 24 hours. Fresh PDA deposition solution was replaced and incubated again at 37°C for 24 hours. Ultrasonic cleaning was then performed to remove unbound PDA, thereby obtaining a titanium alloy fusion device with a PDA coating on its surface.

[0023] S3. preparing a calcium-phosphate deposition solution and performing hydroxyapatite mineralization on the polydopamine coating; Prepare a calcium-phosphate deposition solution: 7mM Ca(NO3)2·4H2O, 4.2mM NaH2PO4·2H2O, and 2mM NaHCO3; place the titanium alloy fusion device with a PDA coating on the surface obtained in step S2 in the calcium-phosphate deposition solution and incubate at 37°C for 12 hours; replace with fresh calcium-phosphate deposition solution and incubate again at 37°C for 12 hours; dry the sample at 37°C to obtain a titanium alloy fusion device with an HA mineralized coating on the surface. Example 2

[0024] Preparation of TBMP2 and construction of bioactive coating S4. Preparation of TBMP2 Construction of expression vector: a recombinant expression vector of TBMP2 was obtained by expressing a (DSS)6 sequence at the N-terminal of the native BMP2 sequence; Transfection expression: the recombinant expression vector was transfected into CHO cells, and cultured for 72 hours; Protein purification: the cell culture supernatant was collected, and purified by nickel column affinity chromatography, hydrophobic chromatography and gel filtration chromatography to obtain TBMP2 protein with a purity of greater than 95%; Activity determination: the ALP activity determination method was used to verify that TBMP2 retained the osteogenic activity of BMP2.

[0025] S5. Grafting TBMP2 protein on the surface of hydroxyapatite mineralized coating A TBMP2 solution with a concentration of 0.5 mg / ml was prepared; the titanium alloy implant with the HA mineralized coating on the surface obtained in step S3 was placed in the TBMP2 solution and incubated at 4°C for 24 hours; after being taken out, it was subjected to freeze-drying treatment and stored for standby, to obtain the bioactive titanium alloy implant with TBMP2 grafted on the surface.

[0026] Test Example 1: In vitro performance characterization of bioactive functional coating 1. Contact angle test The water contact angles of the surfaces of the untreated titanium alloy implant, the titanium alloy implant with the PDA coating on the surface, the titanium alloy implant with the HA mineralized coating on the surface and the bioactive titanium alloy implant with TBMP2 grafted on the surface were measured.

[0027] The results showed that the water contact angle of the untreated titanium alloy implant was 92.5±3.2°, the water contact angle of the titanium alloy implant with the PDA coating on the surface was reduced to 58.7±2.8°, the water contact angle of the titanium alloy implant with the HA mineralized coating on the surface was reduced to 35.6±2.1°, and the water contact angle of the surface of the bioactive titanium alloy implant with TBMP2 grafted on the surface was 29.8±1.9°, and the surface hydrophilicity was significantly improved.

[0028] 2. TBMP2 release behavior The bioactive titanium alloy implant with TBMP2 grafted on the surface was placed in PBS at 37°C, and at the preset time points (1 h, 4 h, 8 h, 1 d, 3 d, 7 d, 14 d, 21 d, 28 d), 100 μL was taken, and the amount of TBMP2 in the sample was detected by ELISA method, for 28 days, and after each sampling, an equal amount of PBS was added to make up the original volume.

[0029] The results show that there is a small initial burst release (about 15%) in the first 8 hours, followed by a steady release phase. The cumulative release reaches about 45% at 14 days and about 65% at 28 days. In comparison, the control group, which has TBMP2 adsorbed directly without HA mineralization, releases about 80% of the protein within the first 24 hours, indicating that the HA mineralization layer has a good sustained release effect on TBMP2.

[0030] 3. Cytotoxicity test MC3T3-E1 cells were cultured in a-MEM complete medium containing 10% fetal bovine serum and 1% double-antibiotic solution at 37°C, 5% CO2 and 95% humidified atmosphere. Cell seeding was performed when the cells reached 80-90% confluence. Cells were seeded in 96-well plates at a density of 2 x 10 4 / ml, 0.1 ml per well. At the same time, the extract of the surface TBMP2 grafted bioactive titanium alloy implant was prepared according to ISO 10993-5, with an extraction ratio of 1.25 cm 2 / ml. After 24 h, the extract of each group was added to the corresponding well. After 1, 3, and 5 days of culture, 10 μL of CCK-8 solution was added to each well, and incubated at 37°C for 1 h. The absorbance was detected at 450 nm. For live / dead cell staining, MC3T3-E1 cells were co-cultured with the extract for 3 days. The cells were stained using a live / dead cell staining kit, and the survival and morphology of the cells were observed under a fluorescence microscope.

[0031] The results show that the cell viability of the TBMP2 grafted group is 101.3 ± 3.1% compared to the control group (normal culture medium). The cell viability is higher than the standard threshold of 70%, indicating that the sample has no cytotoxicity. The live / dead cell staining results show that after 3 days of culture in the extract, the cell morphology is normal and the survival rate is over 95%, further confirming the good cell compatibility of the sample.

[0032] 4. Osteogenic differentiation test MC3T3-E1 cells were seeded in 12-well plates at a density of 2 x 10 4 / mL and cultured with osteogenic induction extract containing sample extract, 10 mM β-glycerophosphate, 0.1 mM dexamethasone, and 50 μg / mL vitamin C. After 7 days of incubation, the cells were lysed using inhibitor-free Western and IP cell lysis solution to extract total protein. The total protein content was determined using a BCA protein quantification kit. ALP activity detection kit was used to detect the ALP activity of MC3T3-E1. In addition, the MC3T3-E1 cells were stained using an ALP staining kit. After 21 days of culture, the MC3T3-E1 cells were stained with alizarin red staining solution. Then, the mineralized nodule was dissolved with 10% cetylpyridinium chloride, and the absorbance was measured at 562 nm.

[0033] The total protein content results show that the total protein content of the cells in the TBMP2 grafted group after 7 days of culture is significantly increased compared to the untreated titanium alloy group. The specific data are: the cell protein content of the untreated group is 143.5 ± 12.6 μg / mL, while that of the TBMP2 grafted group is 198.7 ± 15.3 μg / mL, an increase of about 38.5% (p < 0.05). The ALP test results show that the ALP activity of the TBMP2 grafted group is significantly enhanced after 7 days of culture compared to the untreated titanium alloy group, about 2.8 times that of the untreated group (P < 0.01). The ALP staining results also show that the ALP expression of the osteoblasts in the TBMP2 grafted group is significantly enhanced, with darker staining intensity. Alizarin red staining after 21 days of culture shows that the number and area of mineralization nodules formed in the TBMP2 grafted group are significantly more than those in the untreated group, with a mineralization nodule area about 3.2 times that of the control group (P < 0.01). Quantitative analysis shows that the calcium deposition amount of the TBMP2 grafted group is 3.5 times that of the untreated control group, indicating that the TBMP2 grafted bioactive coating significantly promotes the osteogenic differentiation and mineralization ability of MC3T3-E1 cells.

[0034] 5. Detection of osteogenesis-related gene expression RT-PCR was used to detect the expression of osteogenesis-related genes. MC3T3-E1 cells were seeded in a six-well plate and cultured for 7 days with osteogenic induction extract. Total RNA was extracted and then reverse transcribed into cDNA. Real-time fluorescent quantitative PCR was used to detect the expression of Runx2, ALP, Col1, OCN, OPN and other osteogenesis-related genes.

[0035] The results show that the expression of Runx2, ALP, Col1, OCN and OPN osteogenesis marker genes in the TBMP2 grafted group is significantly up-regulated compared to the untreated titanium alloy group. Among them, Runx2 is up-regulated by 2.6 times, ALP is up-regulated by 3.1 times, Col1 is up-regulated by 2.8 times, OCN is up-regulated by 3.5 times, and OPN is up-regulated by 2.9 times. This indicates that the TBMP2 grafted coating can effectively activate osteogenic transcription factors and promote the expression of osteogenesis-related genes.

[0036] 6. Detection of osteogenesis-related protein expression The expression of osteogenesis-related proteins was detected by Western blot. The MC3T3-E1 cells were cultured in the osteogenic induction extract for 7 days, the total protein of the cells was extracted, and the protein concentration was determined and normalized by BCA kit. The protein samples were heated at 98°C for 5 minutes, loaded onto SDS-PAGE gel and transferred to PVDF membrane. Then, the PVDF membrane was incubated in the blocking solution (5% BSA) for 2 hours. After washing, the PVDF membrane was incubated in the Runx2, ALP, Col1, OCN and OPN antibody solution at 4°C overnight. Subsequently, the PVDF membrane was incubated with the secondary antibody for 2 hours, reacted with the chemiluminescence reagent, exposed and imaged. The Western blot images were semi-quantitatively analyzed by using Image J software.

[0037] The results show that the expressions of Runx2, ALP, Col1, OCN and OPN osteogenesis marker proteins in the TBMP2 grafted group are significantly up-regulated compared with the untreated titanium alloy implant group, which is consistent with the results of osteogenesis gene expression. Semi-quantitative analysis shows that the protein expressions of Runx2, ALP, Col1, OCN and OPN in the TBMP2 grafted group are 2.3 times, 2.8 times, 2.5 times, 3.2 times and 2.7 times of those in the untreated group, respectively, which further confirms that the TBMP2 grafted coating can effectively promote the expression of osteogenesis proteins.

[0038] Test Example 2 Evaluation of the intervertebral fusion and bone integration effects of the bioactive titanium alloy implant in vivo Twelve healthy adult beagles (weight 10-12 kg) were selected and randomly divided into two groups: the control group (n=6) was implanted with untreated 3D printed titanium alloy implants, and the experimental group (n=6) was implanted with bioactive titanium alloy implants grafted with TBMP2 on the surface.

[0039] 1. 3D printing of titanium alloy implants suitable for intervertebral fusion experiments The L4 / L5 vertebrae of beagles were scanned by CT to obtain the parameters of 3D printed implants, which were imported into the 3D printing system to print the implants suitable for the fusion segments of each beagle.

[0040] 2. Construction of bioactive coating The active coating was prepared on the implant according to the methods of Examples 1 and 2 of the present application.

[0041] 3. Establishment of beagle intervertebral fusion model and implantation of implants The beagle dogs were fasted for 12 hours before the operation. The beagle dogs were anesthetized by intramuscular injection of propoxylated phenobarbital + ketamine. Intramuscular injection of 0.5 mg of atropine was performed, and the right lateral position was taken. The left rib arch to the iliac fossa and the lateral thigh were prepared for skin, and routine disinfection was performed. The skin, subcutaneous tissue, external oblique muscle, internal oblique muscle, and peritoneum were sequentially incised, and the peritoneum was separated downward and backward to expose the psoas major muscle and the anterior vertebral body. The surgical site was determined by exploration and puncture, and the important blood vessels around the surgical site were protected. The surgical site was exposed by using an osteoperiosteal elevator, and the L4 / L5 intervertebral disc was removed by exploration and puncture. The cartilage endplate of the upper and lower vertebral bodies was removed by using a curette, and each group of fusion cages was implanted and fixed by using a bone grafting plate. After flushing the wound, the drainage piece was indwelled, and the wound was sutured layer by layer and then disinfected again. Postoperative injection of atropine 0.5 mg and application of antibiotics for 3 days.

[0042] 4. The samples were taken at 6 weeks and 12 weeks after the operation, the L4 / L5 vertebral bodies were completely removed, and Micro-CT scanning was performed to evaluate the intervertebral fusion.

[0043] The results showed that at 6 weeks, the bone volume fraction (BV / TV) of the experimental group was significantly higher than that of the control group (P<0.05); at 12 weeks, the bone volume fraction of the experimental group was further increased and was significantly higher than that of the control group (P<0.01). The number of trabecular bone, the thickness of trabecular bone, and the connectivity of trabecular bone in the experimental group were significantly higher than those in the control group, and the spacing between trabecular bones was significantly smaller than that in the control group (P<0.05).

[0044] 5. Histological observation Hard tissue sections were made from the samples after Micro-CT scanning, and the fusion of the vertebral body and the bone integration of the fusion cage and the surrounding bone tissue were evaluated at the histological level.

[0045] Hard tissue sections and histological staining were performed on the samples after Micro-CT scanning. The results showed that compared with the control group, the density of the newly formed bone tissue around the fusion cage and in the pores in the experimental group was higher, and the bone-fusion cage interface was more compact, with no obvious fibrous tissue gap. At 12 weeks, the fusion cage in the experimental group was basically wrapped by the newly formed bone tissue, and the bone integration effect was significantly better than that in the control group.

[0046] 6. Study on the mechanism of bone integration The fusion cage was carefully removed, and the bone tissue combined with the fusion cage and part of the bone tissue of the upper and lower vertebral bodies in contact with the fusion cage were scraped off for single-cell sequencing to evaluate the mechanism of the fusion cage in promoting intervertebral fusion and bone integration.

[0047] The sequencing results show that, compared with the control group, the gene set related to osteogenesis in the experimental group is significantly enriched, including Runx2, ALP, Col1, OCN, OPN; and the expression of inflammation-related genes TNF-alpha, IL-1beta, IL-6 is down-regulated; the expression of osteoclast differentiation-related gene RANKL is inhibited.

[0048] Finally, it should be noted that: the above-described embodiments only express several embodiments of the present application, and are not used to limit the present application. For those skilled in the art, any modifications, equivalent replacements, improvements, etc. made without departing from the concept of the present application shall be included in the protection scope of the present application. Therefore, the protection scope of the present application patent shall be subject to the appended claims.

Claims

1. A method for preparing an active coating of a surgical titanium alloy fusion device, characterized in that: The following steps are involved: S1. Fabrication of porous titanium alloy fusion cages by 3D printing. S2. Constructing a polydopamine coating on the surface of a titanium alloy fusion cage; S3. preparing a calcium-phosphate deposition solution and performing hydroxyapatite mineralization on the polydopamine coating; S4. TBMP2 protein is grafted onto the surface of the hydroxyapatite mineralized coating to complete the preparation of the active coating of the titanium alloy fusion device.

2. The preparation method according to claim 1, characterized in that The process of constructing the polydopamine coating in step S2 is specifically as follows: dissolving polydopamine in a 30% ethanol / ultrapure water solution to prepare a polydopamine coating deposition solution, placing the titanium alloy fusion device in the polydopamine coating deposition solution, and incubating at 37°C for 24 hours; replacing the deposition solution and incubating again at 37°C for 24 hours; and ultrasonic cleaning to remove unbound polydopamine.

3. The preparation method according to claim 2, characterized in that The concentration of the polydopamine coating deposition solution is 1-4 mg / ml.

4. The preparation method according to claim 1, characterized in that The calcium-phosphorus deposition solution in step S3 specifically includes 6.8-7.5 mM Ca(NO3)2·4H2O, 3.8-4.5 mM NaH2PO4·2H2O and 1.6-2.4 mM NaHCO3.

5. The preparation method according to claim 1, characterized in that The process of performing hydroxyapatite mineralization on the polydopamine coating in step S3 is specifically as follows: placing the fusion device with the polydopamine coating on the surface obtained in step S2 in a calcium phosphate deposition solution and incubating at 37° C. for 12 hours; replacing the calcium phosphate deposition solution and incubating at 37° C. for another 12 hours; and drying at 37° C.

6. The preparation method according to claim 1, characterized in that The process of grafting TBMP2 protein in step S4 is specifically as follows: preparing a TBMP2 solution with a concentration of 0.1-1 mg / ml; placing the mineralized fusion device obtained in step S3 in the TBMP2 solution and incubating it at 4°C for 24 hours; taking it out and freeze-drying it for storage.

7. The preparation method according to claim 1, characterized in that The TBMP2 is a targeted bone morphogenetic protein 2 expressing a (DSS)6 sequence at the N-terminus of the native BMP2 sequence.

8. The preparation method according to claim 1, characterized in that The porosity of the porous titanium alloy fusion device is 30%-70%, and the pore size is 200-600 μm.

9. A 3D printed bioactive titanium alloy fusion device prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the 3D printed bioactive titanium alloy fusion cage according to claim 9 in the preparation of spinal fusion implants, characterized in that: The spinal fusion implant is used for cervical or lumbar intervertebral body fusion.

Images