3D-printed titanium alloy implant with the ability to promote cell proliferation and preparation method and application thereof

Abstract

The application discloses a 3D printing titanium alloy implant with cell proliferation promoting ability and a preparation method and application thereof, and belongs to the technical field of bone repair. The hydrogel provided by the application has a porous structure, can provide nutrient and oxygen transportation, is beneficial to cell penetration and proliferation, and thus promotes the formation of new tissues, provides space for the growth of bones into porous materials; the 3D printing titanium alloy implant gel coating is connected with the 3D printing titanium alloy through hydrogen bonds between amino groups and hydroxyl groups, has compact structure and high bonding stability; the prepared hydrogel has good thermal stability, tensile property, oxidation resistance, antibacterial performance and hemolysis performance, has small swelling property, has excellent degradation performance, has no cytotoxicity, and thus the 3D printing titanium alloy implant is not affected by body temperature and is suitable for bone repair.

Description

Technical Field

[0001] The present invention relates to the technical field of bone repair, and in particular to a 3D-printed titanium alloy implant capable of promoting cell proliferation, and a preparation method and application thereof. Background Art

[0002] 3D printing (3DP), also known as additive manufacturing, is a type of rapid prototyping technology. It uses a 3D model file as the basis and constructs objects layer by layer using bondable materials such as powdered metal or plastic. 3D printing is typically performed using digital material printers and has applications in jewelry, footwear, industrial design, architecture, engineering, and construction (AEC), automotive, aerospace, dental and medical industries, education, geographic information systems, civil engineering, firearms, and other fields.

[0003] Trauma, infection, genetic diseases, and diseases of civilization (obesity, cardiovascular disease) can invade the skeletal muscle system, causing joint damage and affecting the normal function of joints, causing great inconvenience to patients' work and life. Artificial joint replacement has the benefits of alleviating joint pain, maintaining joint mobility, and maintaining joint stability without affecting the total length of the limb. With the advent of an aging society, the number of patients with joint diseases continues to increase, and the number of artificial joint replacement surgeries is also growing. This type of surgery also greatly avoids the pain that patients suffer from facing amputation.

[0004] In recent years, the use of metal implants in medicine has become increasingly widespread, with widespread applications for repairing damaged tissue. Medical titanium alloys, due to their excellent corrosion resistance, mechanical properties, and biocompatibility, are among the most widely used implant materials in orthopedic repair. However, the elastic modulus of traditional dense titanium alloy implants is much higher than that of human bone tissue. The resulting stress shielding effect exposes the surrounding bone to a low stress level for a long time, leading to its gradual absorption and ultimately causing aseptic loosening of the implant.

[0005] The development of 3D printing technology has made it possible to use bioprinted antibacterial hydrogels as porous coatings for the interface fixation of artificial joint prostheses. By 3D printing, a dual-scale porous network structure is constructed, while the hydrogel provides antibacterial protection against postoperative infection around the transplant and an ideal microenvironment for cell adhesion and bone growth, which is conducive to inducing new bone formation and accelerating the bone fusion process. Summary of the Invention

[0006] The purpose of the present invention is to provide a 3D printed titanium alloy implant with the ability to promote cell proliferation, as well as its preparation method and application. By stably connecting hydrogel on the surface of titanium alloy with specific parameters, the purpose of inducing new bone formation and accelerating the bone fusion process is achieved.

[0007] To achieve the above objectives, the present invention provides a method for preparing a 3D-printed titanium alloy implant capable of promoting cell proliferation, comprising the following steps:

[0008] S1, preparation of Mel-AgNPs solution;

[0009] S2, preparing PVA / CS / Mel-AgNPs blend solution;

[0010] S3, preparing 3D printed titanium alloy;

[0011] S4, pre-treating the 3D printed titanium alloy;

[0012] S5. Slowly place the 3D printed titanium alloy processed in step S4 into the PVA / CS / Mel-AgNPs blend solution prepared in step S2 for a certain period of time, freeze it at -20°C and then thaw it. Repeat the freeze-thaw process 5 times to obtain a 3D printed titanium alloy implant with the ability to promote cell proliferation.

[0013] Preferably, the preparation steps of the Mel-AgNPs solution in step S1 are:

[0014] The NaOH solution of black sesame melanin and silver nitrate solution were mixed in a volume ratio of 1:5 and incubated in the dark for 50 minutes. The reaction solution was passed through a 0.22 μm aqueous nanomembrane, and the filtrate was the Mel-AgNPs solution.

[0015] Preferably, the preparation steps of the PVA / CS / Mel-AgNPs blend solution in step S2 are: adding polyvinyl alcohol powder to the acetic acid solution of chitosan, heating to dissolve it, adding the Mel-AgNPs solution prepared in step S1 at 60°C after dissolution, and stirring to mix.

[0016] Preferably, the preparation steps of the 3D printed titanium alloy in step S3 are: firstly perform preliminary design through computer-aided design software, then perform modeling through 3D matic software, and then perform 3D printing to obtain the 3D printed titanium alloy.

[0017] Preferably, when 3D matic modeling is performed, the diameter is set to 4 mm ± 0.5 mm, the depth is set to 7 mm ± 0.5 mm, the pore size is set to 600 μm ± 50 μm, and the porosity is set to 70%.

[0018] Preferably, the pretreatment of the 3D printed titanium alloy in step S4 is as follows: the prepared 3D printed titanium alloy is placed in acetone, anhydrous ethanol, and deionized water in sequence and ultrasonically cleaned twice for 10 minutes each time, and after drying, the 3D printed titanium alloy is immersed in a 5 mol / L, 60°C NaOH aqueous solution for 24 hours, washed with deionized water, and then soaked in deionized water for 8 hours, taken out and immersed in deionized water for ultrasonic cleaning for 15 minutes and then dried.

[0019] Preferably, in step S5, placing the PVA / CS / Mel-AgNPs blend solution prepared in step S2 in the solution for a certain time means standing for 1 hour after bubbles are observed above the solution, freezing means standing at -20°C for 24 hours, and thawing means standing at room temperature for 3 hours.

[0020] A titanium alloy bone repair implant prepared by the preparation method described above.

[0021] A use of the titanium alloy bone repair implant as described above in repairing bone injuries.

[0022] Therefore, the present invention provides a 3D printed titanium alloy implant with the ability to promote cell proliferation, and its preparation method and application, and its specific technical effects are as follows:

[0023] (1) The hydrogel provided by the present invention has a porous structure, which can provide nutrition and oxygen transport, facilitate cell penetration and proliferation, thereby promoting the formation of new tissues and providing space for bone growth into porous materials;

[0024] (2) The 3D printed titanium alloy implant gel coating provided by the present invention is connected to the 3D printed titanium alloy through hydrogen bonds between amino groups and hydroxyl groups, resulting in a compact structure and high bonding stability;

[0025] (3) The hydrogel prepared by the present invention has good thermal stability, stretchability, antioxidant, antibacterial and hemolytic properties, as well as low swelling, excellent degradation performance and no cytotoxicity, so that the 3D printed titanium alloy implant will not be affected by body temperature and is suitable for bone repair.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0028] Figure 1This is the model diagram of the modeling in the first embodiment of the present invention;

[0029] Figure 2 This is the FT-IR spectrum of the PVA / CS / Mel-AgNPs hydrogel in the first test of the present invention;

[0030] Figure 3 The XRD spectrum (a) and crystallinity (b) of the PVA / CS / Mel-AgNPs hydrogel in the second test of the present invention;

[0031] Figure 4 are scanning electron microscope images of the three PVA / CS / Mel-AgNPs hydrogels tested in the present invention, wherein ae are scanning electron microscope images of cross sections with Mel-AgNPs concentrations of 0%, 5%, 10%, 20%, and 50% magnified 500 times, and fj are scanning electron microscope images of cross sections with Mel-AgNPs concentrations of 0%, 5%, 10%, 20%, and 50% magnified 1000 times;

[0032] Figure 5 The DTG curve (a) and TGA curve (b) of the hydrogel measured by the TG (thermogravimetric) method in the fourth test of the present invention are shown;

[0033] Figure 6 This is the result of the investigation on the swelling property of PVA / CS / Mel-AgNPs hydrogel in the fifth test of the present invention;

[0034] Figure 7 This is the result of investigating the degradation performance of PVA / CS / Mel-AgNPs hydrogel in the sixth test of the present invention;

[0035] Figure 8 Figure 1 is the stress-strain curve (a) and mechanical parameter investigation results (b) of PVA / CS / Mel-AgNPs hydrogels with different concentrations in eight experiments of the present invention;

[0036] Figure 9 This is the inhibition zone diagram of the seven 3D printed titanium alloy-PVA / CS / Mel-AgNPs composites tested in the present invention;

[0037] Figure 10 These are scanning electron microscope images of nine 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite materials tested in the present invention; part (a) is a top-down SEM image of the 3D-TC4-coated PVA / CS / Mel-AgNPs hydrogel, and part (b) is a SEM image of the interface between 3D-TC4 and the coated PVA / CS / Mel-AgNPs hydrogel;

[0038] Figure 11This is the result of the antioxidant performance investigation of ten 3D printed titanium alloy-PVA / CS / Mel-AgNPs composites tested in the present invention;

[0039] Figure 12 This is the result of the hemolytic performance investigation of the eleven 3D printed titanium alloy-PVA / CS / Mel-AgNPs composites tested in the present invention;

[0040] Figure 13 These are the results of the cell viability investigation of twelve 3D printed titanium alloy-PVA / CS / Mel-AgNPs composites tested in the present invention. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0042] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0043] This paper uses polyvinyl alcohol (PVA), chitosan (CS), and melanin nanosilver (Mel-AgNPS) as materials, and 3D-printed titanium alloy (pTi) as the implant system. A multifunctional 3D-printed titanium alloy coating composite hydrogel material is prepared by a repeated freeze-thaw method. A series of experiments have shown that the prepared hydrogel has excellent performance as a 3D-printed porous titanium implant and has the characteristics of being suitable for use as a bone repair implant material.

[0044] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0045] Example 1

[0046] A 3D printed titanium alloy implant having the ability to promote cell proliferation is prepared as follows:

[0047] (1) Preparation of Mel-AgNPs solution

[0048] Silver nitrate was used as the raw material and melanin was used as the reducing agent and coating agent to prepare the silver nanoparticle solution in one step. The specific operation was as follows:

[0049] Accurately weigh 10 mg of black sesame melanin powder and dissolve it in 100 mL of 0.1 mol / L NaOH solution to obtain a melanin solution. Accurately weigh 169.87 mg of silver nitrate and dissolve it in distilled water to 1 L to prepare a 1.0 mmol / L AgNO3 solution. Store in the dark (prepare immediately for use). Under constant temperature and magnetic stirring at 60°C (500 r / min), the melanin solution and AgNO3 solution are reacted at a volume ratio of 1:5 at room temperature (20-25°C) in the dark for 50 minutes. The obtained sample is filtered through a 0.22 μm aqueous nanomembrane, and the filtrate is collected to obtain the melanin-nanosilver solution (Mel-AgNPs).

[0050] (2) Preparation of PVA / CS / Mel-AgNPs hydrogel material

[0051] 1 g of chitosan powder was weighed and dissolved in 2% acetic acid solution. The mixture was placed on a stirrer until the chitosan was completely dissolved. After dissolution, 3 g of polyvinyl alcohol powder was added. At the same time, the temperature was raised to 80°C and the mixture was placed on a magnetic stirrer and stirred until it was completely dissolved. Finally, when the temperature dropped to 60°C, 0 mL, 2 mL, 4 mL, 8 mL, and 25 mL of the Mel-AgNPs solution prepared in (1) were added thereto, respectively, so that the concentrations of the Mel-AgNPs solution were 0%, 5%, 10%, 20%, and 50%, respectively. The mixture was placed on a magnetic stirrer and stirred until the Mel-AgNPs were evenly distributed, thereby obtaining five types of PVA / CS / Mel-AgNPs hydrogel materials.

[0052] (3) Preparation of 3D printed titanium alloy

[0053] A. Computer-aided design of 3D-printed titanium alloy (3D-TC4)

[0054] First, through computer-aided design (CAD), the design of printed parts requires commercial software. This software uses Boolean operations on basic primitives, and the component surface is defined as a continuous surface. The surfaces of 3D solid models designed by CAD are represented as continuous surfaces. When converting these surfaces to STL format, the continuous surfaces are represented by triangles. The positions and relationships of these triangles are defined using a specific data structure to ensure that subsequent data processing is time-saving.

[0055] The STL format, a universal standard in the printing industry, is compatible with nearly all 3D printers. Finally, layer processing is performed using QuantAM software. When the print direction is set, the STL 3D solid and the intersecting planes of the continuous z-plane have equal spacing, which represents the thickness of the printed product. When these intersections occur, a 2D sequence of contour lines is obtained. During actual processing, the laser melts all the powder within the contour lines.

[0056] B. 3D matic software modeling

[0057] Referring to the preliminary experimental results, 3D matic software was used to design a titanium alloy prosthetic component with a diameter of 4mm±0.5mm, a depth of 7mm±0.5mm, a pore size of 600μm±50μm, and a porosity of 70%. The model is shown in the figure. Figure 1 .

[0058] C. Pre-treatment of titanium alloy

[0059] ① The prepared 3D printed titanium alloy was placed in acetone, anhydrous ethanol, and deionized water for ultrasonic cleaning twice, each time for 10 minutes, for a total of 1 hour to remove organic matter, oil stains and impurities on the surface of the titanium sheet, and then placed in a 60°C oven for use.

[0060] ② Take the sample treated in ①, immerse it in a 5 mol / L NaOH aqueous solution at 60°C for 24 hours, take out the sample, ultrasonically clean it with deionized water, and then soak it in deionized water at 80°C for 8 hours. Take out the sample and immerse it in deionized water for ultrasonic cleaning for 15 minutes to remove the surface NaOH. Place the sample in a 60°C oven for later use.

[0061] (4) Preparation of 3D printed titanium alloy-PVA / CS / Mel-AgNPs composites

[0062] The treated 3D printed titanium alloy was slowly placed into the five PVA / CS / Mel-AgNPs blend solutions prepared in step (2). After bubbles were observed above the solution, it was allowed to stand for 1 hour. The 3D printed titanium alloy was slowly taken out with pointed tweezers, placed in a small culture dish, and placed in a -20°C refrigerator. After 24 hours, it was taken out, thawed for 3 hours, and placed in a -20°C refrigerator again. After repeated freezing and thawing 5 times according to this process, a 3D printed titanium alloy implant with the ability to promote cell proliferation was obtained: a 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material.

[0063] Experimental Test 1

[0064] The Mel-AgNPs solution prepared in step (2) of Example 1 and the PVA / CS / Mel-AgNPs hydrogel with Mel-AgNPs concentrations of 0%, 5%, 10%, 20%, and 50% were analyzed by Fourier transform infrared spectroscopy (FT-IR). The specific setting for black sesame melanin was as follows: 2 mg of the sample was mixed with 200 mg of KBr to prepare a disk, and the FT-IR was recorded at 4000-500 cm -1 Between, resolution is 4cm-1 FT-IR spectra under 37°C.

[0065] The results are as follows Figure 2 As shown, the stretching vibration absorption peak of hydroxyl (-OH) is at 3428 cm -1 This is consistent with the rich -OH in CS, PVA and melanin. Melanin is at 3396 cm -1 There is also an absorption peak at 3326 cm, indicating that the molecular structure of melanin also contains -OH. By comparison, it is observed that the FT-IR spectrum peak of the synthesized Mel-AgNPs composite is basically consistent with the peak of melanin, indicating that melanin is effectively coated on the surface of the Mel-AgNPs composite during the synthesis process. In addition, the FT-IR spectra of the five PVA / CS / Mel-AgNPs hydrogels with different Mel-AgNPs contents have an absorption peak at 3326 cm -1 NH vibration can be observed at 1457cm -1 There is CH2 shear vibration + CH3 antisymmetric deformation vibration at 1115cm -1 and 745cm -1 CN stretching vibration and NH non-planar vibration can be seen at 3113 cm -1 C=C stretching vibration occurs at 1598cm -1 , 1509cm -1 , 1450cm -1 These results indicate the presence of indole rings in the prepared PVA / CS / Mel-AgNPs hydrogels with different Mel-AgNPs contents. When the Mel-AgNPs complex forms, some peak positions of melanin shift. In Mel-AgNPs, the peaks of aromatic C=C bonds and C=O shift to 1598 cm -1 and 1646cm -1 , indicating that C=O in melanin converts Ag + Reduced to AgNPs. 1388 cm in melanin -1 The absorption peak intensity at 2934 cm was enhanced, which can be inferred that silver interacts with the NH bond on the indole ring of melanin. -1 and 1355cm -1 There are absorption peaks at 1092 cm, corresponding to CH symmetric stretching vibration and in-plane bending vibration, respectively. -1 The peak at 1663 cm is attributed to the stretching vibration of CO. In the CS sample prepared in acetic acid system, a peak at 1663 cm -1 The amide I peak and 1571 cm -1The characteristic absorption peak of -COOH was absent, indicating that during the drying process, most of the unreacted CH3COOH evaporated or reacted with the weakly alkaline solution in the Mel-AgNPs. Due to the superposition of -NH2 and -OH on the carboxyl groups of the melanin silver nanoparticles, the intensity of the -OH stretching vibration peak was observed to increase and shift to higher wavenumbers, inferring that the three substances (CS, PVA, and Mel-AgNPs) are tightly bound together primarily through hydrogen bonding interactions between -NH2 and -OH. This interaction not only enhances the stability of the composite but also may have a positive impact on its properties.

[0066] Experimental Test 2

[0067] The crystal structure of the PVA / CS / Mel-AgNPs hydrogel prepared in Example 1 was analyzed by X-ray diffraction to extract the water supply gel phase. The specific conditions were: room temperature, 2θ range of 5-80°, step length of 1.2° / min, 1s / step. The results are shown in Figure 2. Figure 3 As shown in the figure, part a is the XRD spectrum of PVA / CS / Mel-AgNPs hydrogel, and part b is the crystallinity of PVA / CS / Mel-AgNPs hydrogel. It can be seen that the enhancement of the crystallinity of the cross-linked network of the PVA / CS system is believed to be due to the formation of more hydrogen bonds between Mel-AgNPs and PVA and CS, resulting in a more compact structure.

[0068] Experimental Test Three

[0069] The morphology of the PVA / CS / Mel-AgNPs hydrogel prepared in Example 1 was observed using a scanning electron microscope. Figure 4 As shown, parts ae are scanning electron microscope images of cross sections with Mel-AgNPs concentrations of 0%, 5%, 10%, 20%, and 50% at 500 times magnification, and parts fj are scanning electron microscope images of cross sections with Mel-AgNPs concentrations of 0%, 5%, 10%, 20%, and 50% at 1000 times magnification. Figure 4 It can be seen that the porous structure of these hydrogels can be used to provide nutrient and oxygen transport, allowing cell infiltration and proliferation, thereby promoting the formation of new tissue and providing space for bone to grow into porous materials.

[0070] Experimental Test 4

[0071] The thermal stability of the PVA / CS / Mel-AgNPs hydrogel prepared in Example 1 was investigated by thermogravimetric analysis (TGA). The method was as follows: 10 mg of the hydrogel was placed in a standard aluminum dish and heated at a rate of 10°C / min at 50 cm 3 / min nitrogen flow rate, heating from 30℃ to 600℃. After the test is completed, measure and save the TGA curve, and take its derivative to get the DTG curve, such as Figure 5 As shown, part a is the TGA curve and part b is the DTG curve, which shows that the prepared hydrogel has good overall stability and will not be affected by temperature when used for bone repair implants.

[0072] Experiment Test 5

[0073] The swelling properties of the PVA / CS / Mel-AgNPs hydrogel prepared in Example 1 were investigated as follows:

[0074] Place the hydrogel in a sealed tube containing PBS (pH = 7.4) at 37°C (physiological temperature) to simulate physiological conditions. Take out the hydrogel at regular intervals, wipe the water on the surface of the hydrogel with filter paper, and weigh the hydrogel. Repeat this process until the weight of the hydrogel stabilizes. Calculate the swelling rate of the hydrogel. Repeat four times for each sample and calculate the average value of the results. Use the following formula (1) to calculate the swelling rate:

[0075]

[0076] Where SR is the expansion rate; W t is the mass of the hydrogel at a given time; W0 is the initial mass of the hydrogel.

[0077] The results are as follows Figure 6 As shown in the figure, the swelling properties of PVA / CS / Mel-AgNPs hydrogels are low, less than 25%. This may be due to the formation of microcrystalline structures between CS and PVA molecules during the freezing and thawing process, which are used as crosslinking points to form gels. The double network structure of the gel formed by physical crosslinking has a high crosslinking density, which inhibits the expansion of the gel. On the other hand, it may be due to the NH4 + and COOH form a tight polyelectrolyte complex with Mel-AgNPs through ionic interactions, which greatly limits its expansion capacity. The higher the Mel-AgNPs content, the tighter the hydrogel network structure, the tighter the internal pore structure, the smaller the constraint on the free movement of the hydrogel skeleton, and the lower the water expansion rate. For synthetic articular bone and cartilage materials, no swelling or low swelling in physiological solutions is crucial. If the hydrogel swells too much after implantation, it will cause unnecessary pressure on the surrounding tissues and cause secondary damage. Therefore, low-swelling hydrogels can be used as implants inside the pores of 3D-printed titanium alloys for bone repair.

[0078] Experimental Test 6

[0079] The degradation performance of the PVA / CS / Mel-AgNPs hydrogel prepared in Example 1 was investigated by an in vitro degradation experiment as follows:

[0080] PVA / CS / Mel-AgNPs hydrogels containing different Mel-AgNPs concentrations were cut into cylinders with a diameter of 10 mm and a thickness of 5 mm. Each hydrogel sample was weighed (denoted as W1), and a phosphate buffer solution (PBS, pH 7.4) containing 40 U / mL cellulase was added to cover the sample. The sample was incubated at 37 ° C and 100 r / min, and the PBS buffer was replaced every 24 h. After the incubation, the sample was rinsed with deionized water, dried at 60 ° C and weighed (denoted as W2). Each group was repeated three times. The degradation rate was calculated using the following formula (2):

[0081]

[0082] Where W1 is the initial mass of the hydrogel; W2 is the weight after immersion in PBS buffer.

[0083] The results are as follows Figure 7 As shown, the degradation rate of the hydrogel can reach more than 70% within a certain period of time after being implanted in the body.

[0084] Experimental Test 7

[0085] The stretchability of the PVA / CS / Mel-AgNPs hydrogel prepared in Example 1 was investigated as follows:

[0086] The specimens were cut into dumbbell shapes with an initial gauge length of 12 ± 0.5 mm and a width of 2.5 ± 0.2 mm. Tensile tests were performed at room temperature (25°C) at a rate of 20 mm / min. The corresponding fracture stress σb and fracture strain εb were extracted from the stress-strain curves of at least three individual tests.

[0087] The results are as follows Figure 9 Part (a) shows the stress-strain curves of hydrogels at different concentrations. All hydrogels exhibited a certain degree of stretchability, breaking at strains between 1.5% and 2%. The maximum stress reached nearly 0.5 MPa at a Mel-AgNPs concentration of 10%. The mechanical properties of the hydrogels decreased with increasing Mel-AgNPs concentration. Due to the presence of -OH groups in the Mel-AgNPs solution, the overall PVA / CS concentration decreased, leading to lower acidity and fewer hydrogen bonds. However, the hydrogels still maintained a certain level of mechanical properties.

[0088] As can be seen from part b, the overall elongation at break of hydrogels with different concentrations are 173.54%, 178.06%, 175.63%, 168.16% and 157.94%, respectively. This shows that the hydrogel has strong elongation and deformation capabilities. When the hydrogel is subjected to the maximum load, the elastic modulus of hydrogels with different concentrations can be used as an indicator to measure the ease of elastic deformation of the material. The value of the elastic modulus represents the stress that causes a certain elastic deformation of the material. The measured values ​​of the elastic modulus of hydrogels with different concentrations in the figure are 1.74MPa, 1.78MPa, 1.76MPa, 1.58MPa and 1.59MPa, respectively. Therefore, when the gel is injected into the metal, the gel is not prone to elastic deformation and will not affect the performance of the composite material. Therefore, the prepared hydrogel is very suitable for bone defect repair implants.

[0089] Experimental Test 8

[0090] The antibacterial properties of the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material prepared in Example 1 were investigated using the disk diffusion method, as follows:

[0091] The composite material was tested under normal light and near infrared light (808 nm, 5 W / cm 2 , 10 min) after enhanced in vitro antibacterial performance, the disc diffusion method was used to determine the concentration of 1.0×10 5 100 μL of a bacterial suspension in the logarithmic growth phase at a CFU / mL was added to the surface of solidified LB solid medium and evenly spread with an applicator. The mixture was allowed to stand at room temperature for 5 minutes. A sterilized 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite was applied to a culture dish and incubated in a 37°C incubator for 24 hours. The diameter of the inhibition zone (mm) was observed and measured. Results are expressed as x ± s. All experiments were performed in triplicate.

[0092] The results are as follows Figure 9 As shown, the blue circles indicate natural light exposure, while the red circles indicate near-infrared exposure. The 3D-printed titanium alloy material produced no inhibition zones against either E. coli or S. aureus, while the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs prepared in Example 1 produced inhibition zones against both E. coli and S. aureus. This indicates that the antibacterial components of the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs primarily come from the antibacterial hydrogel in the coating. Furthermore, the inhibition zones produced by the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs against E. coli and S. aureus were concentration-dependent: higher concentrations of Mel-AgNPs resulted in larger inhibition zones.

[0093] Experimental Test Nine

[0094] The morphology of the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite material prepared in Example One was observed by scanning electron microscopy, and the results are shown in Figure 8, where (a) is a 3D-printed titanium alloy-PVA / CS / Mel-AgNPs overhead SEM, and the hydrogel can be seen to have filled the 3D-printed titanium alloy. (b) is an SEM of the contact site between the 3D-printed titanium alloy and the hydrogel in the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite material. As can be clearly seen in the figure, the hydrogel still has a porous layered structure, which is conducive to promoting the distribution of nutrients and providing a good environment for cell adhesion and growth. Figure 10

[0095] Ten samples were tested in the experiment

[0096] The antioxidant properties of the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite material prepared in Example One were investigated, as follows:

[0097] (1) ABTS free radical scavenging activity

[0098] ABTS+ free radical scavenging experiment: ABTS and potassium persulfate solution were mixed and left in the dark for more than 12 h to prepare an ABTS ion solution. Before use, the ABTS+ solution was adjusted to an absorbance value of 0.7 ± 0.02 at 734 nm with ethanol. After adding the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite material and reacting for a period of time, the scavenging capacity of each 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite material was calculated according to the absorbance of the solvent at 734 nm, and the ABTS+ free radical scavenging rate of each group was calculated using formula (3). Each group of experiments was repeated an average of 3 times.

[0099]

[0100] In the formula, A1 is the absorbance value after the ABTS free radical reacts with the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite material; A2 is the absorbance value of the reaction product of anhydrous ethanol and ABTS; and A0 is the initial absorbance of the ABTS free radical solution.

[0101] (2) DPPH free radical scavenging activity

[0102] ​The antioxidant activity of the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite was determined by scavenging 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH). A certain amount of DPPH was weighed and dissolved in anhydrous ethanol to prepare a 1 mmol / L DPPH solution. The solution was stored in the dark and the absorbance (A0) was measured at 517 nm. An 8 mm diameter 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite was immersed in the DPPH solution, mixed thoroughly, and placed in the dark for 30 minutes. The absorbance (A1) was measured at 517 nm. The 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite was immersed in anhydrous ethanol solution as a control, and the absorbance (A2) was measured at 517 nm. Each experiment was repeated three times and the average was taken. The scavenging rate of the hydrogel for DPPH free radicals was calculated according to formula (4).

[0103]

[0104] Where A1 is the absorbance value of the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material after the reaction with DPPH free radicals; A2 is the absorbance value of the control group after the reaction with anhydrous ethanol and DPPH free radicals; A0 is the initial absorbance value of the DPPH free radical solution.

[0105] The results are as follows Figure 11 As shown, the 3D-printed titanium alloy without hydrogel coating has very weak antioxidant capacity, demonstrating that 3D-TC4 itself has almost no antioxidant activity. However, the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composite prepared in Example 1 has high antioxidant capacity. The ABTS and DPPH free radical scavenging rates of 3D-printed titanium alloy implants coated with PVA / CS / Mel-AgNPs hydrogel were as high as 90.87% and 85.67%, respectively, demonstrating the important role of the antibacterial hydrogel in enhancing overall antioxidant activity.

[0106] Test 11

[0107] The hemolytic performance of the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material prepared in Example 1 was investigated as follows:

[0108] Red blood cells were obtained by centrifuging mouse blood at 1000 rpm for 10 minutes. The red blood cell suspension was diluted to 2% (v / v) with normal saline, and the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material and the diluted blood were centrifuged at 37°C (1000 rpm, 2 minutes) and incubated for 1 hour. After centrifugation, the supernatant was transferred to a 96-well plate, and the absorbance of the supernatant was read at 540 nm using a microplate reader. In addition, distilled water was used as a positive control and normal saline was used as a negative control. Each experiment was repeated three times. The hemolysis rate was calculated using the following formula (5):

[0109]

[0110] Where Ap is the absorbance value of the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material group; At is the absorbance value of the positive control distilled water; Ab is the absorbance value of the negative control physiological saline.

[0111] The results are as follows Figure 12 As shown in the figure, the hemolysis rate of all 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite materials is less than 5%, indicating that the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite scaffold system has good blood compatibility.

[0112] Test 12

[0113] The cytotoxicity of the 3D printed titanium alloy-PVA / CS / Mel-AgNPs composite material prepared in Example 1 was investigated as follows:

[0114] The viability of L929, MC3T3-E1, and MCF-7 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hydrogels (8 mm in diameter) were placed in 24-well plates (the hydrogels were disinfected with 75% ethanol for 30 minutes, exposed to ultraviolet light for 30 minutes, and washed twice with PBS). L929, MC3T3-T1, and MCF-7 cell suspensions (4 × 10 5 cells / mL) were inoculated into each well and cultured for 24 h. The culture medium was then aspirated, and 360 μL of MTT solution was added to each well. After 4 h, the culture medium was removed again, and 360 μL of dimethyl sulfoxide (DMSO) was added. The cells were thoroughly mixed on a shaker for 5 min, then transferred to a 96-well plate. The absorbance at 570 nm was measured using a microplate reader. The cell survival rate and inhibition rate of the 3D-printed titanium alloy-PVA / CS / Mel-AgNPs composites at different concentrations of Mel-AgNPs were calculated according to Equation (6).

[0115]

[0116] Where Ai is the treatment group with different concentrations of hydrogel culture medium added; A0 is the treatment group without hydrogel.

[0117] The results are as follows Figure 13 As shown, the L929 cell survival rate of 3D-TC4, used as a control, was 83.13%, demonstrating that the 3D-TC4 material is non-toxic to cells. In the context of a 3D-TC4 scaffold coated with PVA / CS / Mel-AgNPs hydrogel, the L929 cell survival rates were 89.68%, 91.64%, 92.13%, 94.31%, and 92.03% at Mel-AgNPs concentrations of 0%, 5%, 10%, 20%, and 50%, respectively. This demonstrates that the 3D-TC4 scaffold coated with PVA / CS / Mel-AgNPs hydrogel is non-toxic to cells and that a 20% Mel-AgNPs concentration is most beneficial for cell growth.

[0118] Therefore, the hydrogel provided by the present invention has a porous structure, which can provide nutrition and oxygen transport, facilitate cell penetration and proliferation, thereby promoting the formation of new tissues and providing space for bones to grow into porous materials; the provided 3D printed titanium alloy implant gel coating is connected to the 3D printed titanium alloy through hydrogen bonds between amino groups and hydroxyl groups, with a compact structure and high binding stability; the prepared hydrogel has good thermal stability, stretchability, antioxidant, antibacterial and hemolytic properties, while having low swelling, excellent degradation performance and no cytotoxicity, so that the 3D printed titanium alloy implant will not be affected by body temperature and is suitable for bone repair.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a 3D printed titanium alloy implant capable of promoting cell proliferation, characterized in that: Here are the steps: S1, preparation of Mel-AgNPs solution; S2, preparing a PVA / CS / Mel-AgNPs blend solution, wherein PVA is polyvinyl alcohol; CS is chitosan; S3, preparing 3D printed titanium alloy; S4, pre-treating the 3D printed titanium alloy; S5. Slowly placing the 3D printed titanium alloy processed in step S4 into the PVA / CS / Mel-AgNPs blend solution prepared in step S2 for a certain period of time, freezing at -20°C and then thawing, repeating the freeze-thaw process 5 times to obtain a 3D printed titanium alloy implant with the ability to promote cell proliferation; The preparation steps of the Mel-AgNPs solution in step S1 are: The NaOH solution of black sesame melanin and silver nitrate solution were mixed in a volume ratio of 1:5 and incubated in the dark for 50 minutes. The reaction solution was passed through a 0.22µm aqueous nanomembrane, and the filtrate was the Mel-AgNPs solution.

2. The method for preparing a 3D printed titanium alloy implant having the ability to promote cell proliferation according to claim 1, characterized in that: The preparation steps of the PVA / CS / Mel-AgNPs blend solution in step S2 are as follows: adding polyvinyl alcohol powder to the acetic acid solution of chitosan, heating to dissolve it, adding the Mel-AgNPs solution prepared in step S1 at 60°C after dissolution, and stirring to mix.

3. The method for preparing a 3D printed titanium alloy implant having the ability to promote cell proliferation according to claim 1, characterized in that: The preparation steps of the 3D printed titanium alloy in step S3 are: firstly perform preliminary design using computer-aided design software, then perform modeling using 3D matic software, and then perform 3D printing to obtain the 3D printed titanium alloy.

4. The method for preparing a 3D-printed titanium alloy implant capable of promoting cell proliferation according to claim 3, characterized in that: When 3D matic modeling was used, the diameter was set to 4 mm ± 0.5 mm, the depth was 7 mm ± 0.5 mm, the pore size was 600 μm ± 50 μm, and the porosity was 70%.

5. The method for preparing a 3D printed titanium alloy implant capable of promoting cell proliferation according to claim 1, characterized in that: In step S4, the 3D printed titanium alloy is pretreated as follows: the prepared 3D printed titanium alloy is placed in acetone, anhydrous ethanol, and deionized water in sequence and ultrasonically cleaned twice for 10 minutes each time. After drying, the 3D printed titanium alloy is immersed in a 5 mol / L, 60°C NaOH aqueous solution for 24 hours. After washing with deionized water, it is soaked in deionized water for 8 hours, taken out and immersed in deionized water for ultrasonic cleaning for 15 minutes and then dried.

6. The method for preparing a 3D-printed titanium alloy implant capable of promoting cell proliferation according to claim 1, characterized in that: In step S5, placing the PVA / CS / Mel-AgNPs blend solution prepared in step S2 for a certain time means standing it for 1 hour after bubbles are observed above the solution, freezing means placing it at -20°C for 24 hours, and thawing means placing it at room temperature for 3 hours.

7. A titanium alloy bone repair implant prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the titanium alloy bone repair implant according to claim 7 in the preparation of bone damage repair materials.

Images