Titanium implant multifunctional composite coating with anti-active oxygen and anti-inflammatory dual performance and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a titanium implant multifunctional composite coating with anti-active oxygen and anti-inflammatory dual performance and a preparation method of the titanium implant multifunctional composite coating. The coating is composed of a lanthanum oxide (La2O3) nano-particle layer growing on the surface of the titanium implant in situ and queen lily glycoside A (RA) loaded on the lanthanum oxide (La2O3) nano-particle layer and can continuously release La < 3 + > and RA. The preparation method comprises the following steps: performing alkali heat treatment on the titanium substrate; a La (OH) 3 coating is synthesized on the surface in situ through a hydrothermal reaction; the La2O3 nano-particles are calcined and converted into a La2O3 nano-particle layer with a porous structure; and finally loading RA through an impregnation method. The coating can effectively remove ROS, regulate immune inflammatory response, promote osteogenic differentiation and inhibit osteoclast activity, thereby remarkably promoting osseointegration. The problem of osseointegration under the osteoporosis pathological microenvironment is effectively solved, the preparation process is simple and controllable, and the composition is suitable for bone defect repair of osteoporosis patients and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a titanium implant multifunctional composite coating with dual properties of anti-active oxygen and anti-inflammation and a preparation method thereof. BACKGROUND

[0002] Osteoporosis, as a systemic bone metabolism disease, is characterized by bone mass loss, microstructure degradation and mechanical property decline in the macroscopic level, which leads to a significant increase in the risk of bone fracture. In the micro-mechanism level, the disease presents multi-dimensional characteristics, such as pH value reduction, abnormal ROS level increase and chronic inflammation state in the microenvironment level; mesenchymal stem cell osteogenic differentiation ability attenuation, osteoclast activity hyperactivity and macrophage polarization imbalance (M1 type dominant) in the cell metabolism level. This multi-level homeostasis disorder leads to the osteoporotic bone defect repair cycle to be prolonged to more than 3 months, far exceeding the normal bone tissue healing speed (usually <2 months).

[0003] Titanium and titanium alloys have become the mainstream implant materials for osteoporotic bone defect repair due to their excellent mechanical strength, corrosion resistance and biological safety. However, their inherent biological inert surface characteristics seriously restrict the integration efficiency of the implant and the host bone tissue. In the pathological microenvironment of osteoporosis, the abnormal increase in osteoclast activity and the inhibited osteogenic ability form a vicious cycle, which leads to the formation of the bone integration interface around the implant to be blocked, and significantly increases the incidence of early postoperative loosening and displacement. More seriously, oxidative stress and chronic inflammation in the pathological microenvironment further inhibit the new bone formation process by destroying the intercellular network (including cytokine signaling, material-cell interface interaction and cell-cell interaction). Clinical data shows that the bone implant surgery failure rate of osteoporosis patients is about 40% higher than that of healthy people, which highlights the urgent need to improve the bone integration performance under pathological conditions.

[0004] It is worth noting that recent studies have found that by adjusting the microenvironment homeostasis (such as inhibiting oxidative stress and regulating inflammation level), the osteoporosis process can be effectively reversed and bone regeneration can be promoted. However, these strategies are often single-functioned, and it is difficult to synergistically cope with the multiple obstacles such as oxidative stress, inflammation, osteogenesis inhibition and osteoclast hyperactivity in the osteoporosis microenvironment.

[0005] In recent years, rare earth oxides such as lanthanum oxide (La2O3) have attracted attention due to their good biocompatibility, controllable ion release, and abundant mesoporous structure, as well as their potential osteogenic induction and osteoclast inhibition capabilities. Meanwhile, active ingredients extracted from natural plants, such as Regaloside A (RA), have been proven to have excellent antioxidant and anti-inflammatory properties. However, there is currently no technology that integrates both on the surface of titanium implants to create a multifunctional composite coating that can simultaneously address the multiple challenges of oxidative stress, chronic inflammation, osteogenesis inhibition, and excessive osteoclast activity in the pathological microenvironment of osteoporosis. Therefore, developing a titanium implant composite coating that integrates the advantages of both is of great significance in significantly improving the bone repair effect and long-term stability of implants under osteoporotic pathological conditions. SUMMARY

[0006] The purpose of the present application is to provide a titanium implant multifunctional composite coating with dual antioxidant and anti-inflammatory properties and a preparation method thereof, which can effectively remove ROS, regulate immune inflammatory response, promote osteogenic differentiation and inhibit osteoclast activity under osteoporotic pathological conditions, thereby significantly promoting osseointegration.

[0007] To achieve the above-mentioned purpose, in one aspect, the present application provides a titanium implant multifunctional composite coating, which is formed in situ on the surface of a titanium or titanium alloy implant and comprises a La2O3 nanoparticle layer and Regaloside A loaded on the La2O3 nanoparticle layer.

[0008] In another aspect, the present application also provides a preparation method of a titanium implant multifunctional composite coating, comprising the following steps: S1. Alkaline heat treatment of a titanium or titanium alloy substrate to obtain a titanium implant surface AT; S2. In situ synthesis of a La(OH)3 coating on the AT through a hydrothermal reaction to obtain AT / La(OH)3; S3. Calcination of the AT / La(OH)3 to convert La(OH)3 to La2O3 to obtain AT / La2O3; S4. Immersion of the AT / La2O3 in a Regaloside A solution to load RA in the porous structure of the coating to obtain an AT / La2O3 / RA composite coating.

[0009] Further, in S1, the alkaline heat treatment is to ultrasonically wash titanium discs and titanium rods with pure titanium in a 4-6 M NaOH solution at 70-90°C for 24 hours after ultrasonic washing with ethanol and deionized water for 1 hour each.

[0010] Further, in S2, the hydrothermal reaction is that the AT is placed in a 10 mL hydrothermal reaction solution composed of urea and La(NO3)3·6H2O in a molar ratio of 1:1, and the reaction kettle is heated at 100-120 DEG C for 12 hours.

[0011] Further, in S3, the calcination temperature is 600-800 DEG C, and the time is 2 hours.

[0012] Further, in S4, the concentration of the oxymatrine solution is 50-200 μg / mL.

[0013] The application further provides application of the titanium implant multifunctional composite coating in preparation of an implant material for repairing osteoporotic bone defects.

[0014] Further, the titanium implant multifunctional composite coating continuously releases La 3+ and oxymatrine.

[0015] Further, the titanium implant multifunctional composite coating has antioxidant activity and anti-inflammatory effect, promotes proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs), and inhibits differentiation of osteoclasts.

[0016] Further, the titanium implant multifunctional composite coating is used for repairing bone defects of osteoporosis patients, and promotes new bone formation and osseointegration.

[0017] The titanium implant multifunctional composite coating with dual properties of anti-reactive oxygen and anti-inflammation and the preparation method thereof have the following beneficial effects: (1) multifunctional synergistic effect: La2O3 with osteogenesis / osteoclast regulation ability and RA with strong antioxidant / anti-inflammatory ability are combined to construct a multifunctional coating. 3+ The synergistic release of La 3+ and RA can simultaneously intervene in multiple key factors (high ROS, chronic inflammation, and osteogenesis inhibition) of the pathological microenvironment of osteoporosis.

[0018] (2) excellent antioxidant performance: the loaded RA can effectively remove excess ROS in the pathological microenvironment, relieve oxidative stress, and create favorable conditions for bone regeneration.

[0019] (3) effective immune regulation and anti-inflammatory effect: the coating can promote the polarization of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, up-regulate anti-inflammatory factors, and down-regulate pro-inflammatory factors, thereby reducing the inflammatory response around the implant.

[0020] (4) strong osteogenesis promotion and osteoclast inhibition: the continuously released La 3+ can directly promote osteogenic differentiation of MSCs and up-regulate expression of osteogenesis-related genes; meanwhile, La 3+Both RA and AT can effectively inhibit the differentiation and activity of osteoclasts, and down-regulate the expression of osteoclast-related genes.

[0021] (5) Improve bone integration: in the osteoporosis animal model, the coating of the present application significantly reduces the ROS level and inflammatory response around the implant, promotes the formation of a large number of mature new bones, increases the bone volume fraction and the number of trabeculae, and exhibits excellent in vivo bone integration ability.

[0022] (6) Simple and controllable preparation process: the present application uses conventional methods such as hydrothermal, calcination and immersion, and the process is simple, reproducible and easy to realize large-scale production.

[0023] The technical solutions of the present application will be further described in detail below with the help of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure is a schematic diagram of the design strategy of AT / La2O3 / RA for removing reactive oxygen species (ROS) in the osteoporosis microenvironment, regulating immune response and highlighting the potential of promoting bone integration; Figure 2 Figure A is a scanning electron microscope (SEM) image and an energy dispersive spectroscopy (EDS) image of all samples (white scale bars are 10 μm and 50 μm, respectively), B is an X-ray diffraction (XRD) analysis of AT / La(OH)3 and AT / La2O3, C is the water contact angle of different samples, D is the cumulative release amount of La 3+ in AT / La(OH)3, AT / La2O3 and AT / La2O3 / RA, E is the cumulative release amount of RA in AT / La2O3 / RA; Figure 3 Figure A is the H2O2 scavenging ability of all coating samples, B is the total antioxidant capacity analysis of all samples, C is the cell live and cytoskeleton staining results of MSCs after being treated with different samples for 7 days (scale bar: 20 μm), D is the quantitative analysis of MSCs proliferation after being treated with different samples for 1 day, E is the quantitative analysis of MSCs proliferation after being treated with different samples for 3 days, F is the quantitative analysis of MSCs proliferation after being treated with different samples for 7 days; Figure 4 Figure is the cytoskeleton staining diagram of MSCs of different samples; Figure 5Image A shows the live and dead cell staining of RAW264.7 cells on different sample surfaces; image B shows the viability of RAW264.7 cells after 1 and 3 days of culture on different sample surfaces; images C and D show the mRNA expression levels of related inflammatory factors in RAW264.7 cells after 1 day of culture on different sample surfaces; and image E shows the CD86 and CD206 fluorescence staining images of RAW264.7 cells on different sample surfaces. Error bars represent mean ± standard deviation. Sample size n=3. p<0.05, p<0.01; Figure 6 This is a quantitative immunofluorescence analysis of CD86 and CD206. Figure 7 In the table, A and B represent the expression levels of osteogenic-related genes after 3 and 7 days of MSC culture, C represents the ALP staining and Alizarin Red staining results after 7 and 14 days of MSC culture, D represents the ALP activity after 7 days of MSC culture, and E represents the mineralization capacity of MSC culture after 7 and 14 days. Figure 8 Table A shows the collection and application process of conditioned medium; Table B shows the proliferation of MSCs at 1, 3, and 7 days; Tables C, D, and E show the staining results and quantitative analysis of ALP activity and mineralization in MSCs under different conditioned medium treatments; Tables F and G show the expression levels of osteogenic-related genes in MSCs after 3 and 7 days of treatment with different conditioned mediums. Error bars represent the mean ± standard deviation, and the sample size n=3. p<0.05 p<0.01; Figure 9 In Figure A, the expression levels of osteoclast-related genes (TRAP, c-FOS, CTSK, and NFATc1) in surface cells of different samples are shown. In Figure B, the TRAP activity of surface cells of different samples after 1 day of culture is shown. In Figure C, CLSM images of multinucleated cells on the surfaces of different materials are shown. Scale bar: 20 μm. Error bars represent mean ± standard deviation. Sample size n=3. p<0.05 p<0.01.

[0025] Figure 10 Image A shows frozen bone sections stained with DHE from different samples, displaying ROS fluorescence intensity (GP: growth plate, BM: bone marrow, TB: trabeculae). Images B and C are histological images of bone sections stained with HE and Masson trichrome. Figure 11A is the new bone formation after 2 months of surgical implantation, B is the quantitative statistical result of bone volume fraction (BV / TV), C is the quantitative statistical result of trabecular number (Tb.N), D is the quantitative statistical result of trabecular space (Tb.Sp), p<0.01, . DETAILED DESCRIPTION

[0026] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0027] Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods not specified in the following examples are generally determined according to national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.

[0028] Unless otherwise defined or specified, all professional and scientific terms used in the present application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0029] The present application is based on the characteristics of the pathological microenvironment of osteoporosis and designs a coating of lanthanum oxide nanoparticles loaded with RA (as shown in Figure 1 ).

[0030] Example 1: Preparation of composite layer: After ultrasonic washing with ethanol and deionized water for 1 h, the pure titanium containing titanium disc and titanium rod was reacted in a NaOH solution (5 M) at 80°C for 1 day, and the treated sample was named AT (alkali heat treatment). The AT sample was heated at 105°C for 12 h in a reaction kettle of 10 mL hydrothermal reaction solution composed of 21.6 mM urea and La(NO3)3·6H2O, and the treated sample was named AT / La(OH)3. Then, the obtained sample was calcined in a muffle furnace at 750°C for 2 h, and the prepared sample was named AT / La2O3. Finally, the calcined sample was immersed in a 100 μg / mL radix rehmanniae preparata solution overnight to obtain the final sample and was named AT / La2O3 / RA.

[0031] Example 2: Characterization of example 1 sample: (1) The surface morphology of all samples was characterized by SEM detection. As shown in Figure 2As shown in FIG. 1A, the pure Ti sample shows a relatively smooth surface morphology, and the other groups can observe relatively rough nanostructures. Under a higher magnification, AT presents a lot of nano-scale cross grooves. The AT / La(OH) 3 coating surface can observe smaller needle-like and shuttle-like nanoparticle structures, and the AT / La 2 O 3 surface is more of larger shuttle-like nanoparticle structures. When La(OH) 3 is converted into La 2 O 3 by calcination, as the temperature continues to rise, the grain boundary interface energy is higher, which is more conducive to grain boundary migration, that is, the faster the grain growth rate, the larger the particle size. In addition, the pore size in the particle will also increase when the metal hydroxide is converted into oxide, so the porous internal structure of La(OH) 3 after being converted into La 2 O 3 is more conducive to loading RA. As shown in FIG. 1B, the surface of the AT / La 2 O 3 / RA coating is blurred compared with the AT / La 2 O 3 coating, because the La 2 O 3 nanoparticles load RA to form a film-like structure on the coating surface, thereby proving that the wubanglycoside has been successfully coated. Figure 2 As shown in FIG. 1C, compared with the control group, a large amount of La appeared in the AT / La(OH) 3, AT / La 2 O 3 and AT / La 2 O 3 / RA groups, and the content reached more than 50%, in addition, a large amount of C element appeared in the AT / La 2 O 3 / RA, and the content was 27%, which mainly came from RA, which also proved that RA has been successfully loaded in the coating. Figure 2 As shown in FIG. 1D, compared with the control group, a large amount of La appeared in the AT / La(OH) 3, AT / La 2 O 3 and AT / La 2 O 3 / RA groups, and the content reached more than 50%, in addition, a large amount of C element appeared in the AT / La 2 O 3 / RA, and the content was 27%, which mainly came from RA, which also proved that RA has been successfully loaded in the coating.

[0032] (2) The crystal phase of various samples was analyzed by XRD, as shown in FIG. 2A, the characteristic peaks of the corresponding substances were shown in the samples of AT / La(OH) 3 and AT / La 2 O 3, which indicated that the La(OH) 3 and AT / La 2 O 3 were successfully synthesized in situ on the surface of the AT / La(OH) 3 and AT / La 2 O 3 samples. Figure 2 As shown in FIG. 2B, the characteristic peaks of the corresponding substances were shown in the samples of AT / La(OH) 3 and AT / La 2 O 3, which indicated that the La(OH) 3 and AT / La 2 O 3 were successfully synthesized in situ on the surface of the AT / La(OH) 3 and AT / La 2 O 3 samples.

[0033] (3) The hydrophilicity and hydrophobicity of the surface of the biomaterial is also one of the key factors for regulating cell behavior. Figure 2 As shown in FIG. 2C, the hydrophilicity and hydrophobicity of the surface of each sample was detected by using a water contact angle instrument, and the results showed that the water contact angles of Ti, AT, AT / La(OH) 3, AT / La 2 O 3 and AT / La 2 O 3 / RA were 57.3°, 46.9°, 17.7°, 16.63° and 15.49° respectively, it can be seen that the water contact angle of the surface coating of the experimental group is obviously smaller than that of the pure titanium surface, which indicates that the hydrophilicity of the titanium surface is greatly improved after functional modification, which will be conducive to the adhesion of MSCs on the material surface.

[0034] (4) To determine the ion release of the sample in an acidic microenvironment, the sample was immersed in 5 mL of physiological saline (pH 5.8) while standing (simulating the slightly acidic environment of osteoporosis); after incubation for the corresponding time, the soaking solution was removed, and fresh solution was added at 37°C at different time intervals (0, 1, 3, 7, 11, 14 and 21 days); finally, the collected liquid was centrifuged at high speed (4°C, 12000 rpm, 10 min), and La was detected by inductively coupled plasma atomic emission spectrometry (ICP-AES) and ultraviolet absorption spectrometry, respectively. 3+ The concentrations of ions and lily glycosides. For example... Figure 2 As shown in Figures D and E, the three groups AT / La(OH)3, AT / La2O3, and AT / La2O3 / RA can all release La relatively stably and continuously. 3+ The release time was 28 days, with cumulative release amounts of 5.677 mg / L, 8.614 mg / L, and 5.972 mg / L on day 28, respectively. Compared to the AT / La(OH)3 and AT / La2O3 groups, the AT / La2O3 / RA coating showed higher La release levels in the first 7 days. 3+ The release behavior exhibited a significant slow-release phenomenon, with the lowest release amount among the three groups. Furthermore, the release rates of the AT / La₂O₃ and AT / La₂O₃ / RA groups were slightly lower than that of the AT / La(OH)₃ group, indicating that the structures of the first two coating groups were more stable than the latter. This also proves that converting La(OH)₃ to La₂O₃ and introducing RA can effectively improve the stability of the coating. Figure 2 As shown in Figure E, the RA release curve of the AT / La2O3 / RA group shows that the release rate of RA can reach 70% of the total amount in the first 7 days, which is beneficial for the immediate inflammatory regulation after material implantation.

[0035] (5) Next, the in vitro antioxidant capacity of the coating was evaluated. First, the H2O2 scavenging capacity of each group of samples was tested using an H2O2 scavenging kit, and the results are as follows: Figure 3 As shown in Figure A, approximately 60% of the H2O2 in the AT / La2O3 / RA group was decomposed within 48 hours, while the total H2O2 concentration in the Ti, AT, AT / La(OH)3, and AT / La2O3 groups showed no significant change. Therefore, it can be determined that the addition of RA significantly improves the H2O2 scavenging ability of the materials. Further analysis using a FRAP kit revealed the total antioxidant capacity of each group of samples, with results shown below. Figure 3 As shown in Figure B, AT / La2O3 / RA exhibits strong overall antioxidant capacity, while the overall antioxidant capacity of the Ti, AT, AT / La(OH)3, and AT / La2O3 groups is almost negligible. Therefore, the AT / La2O3 / RA coating possesses excellent antioxidant capacity.

[0036] In summary, the above illustrates that the AT / La2O3 / RA coating can release RA preferentially in the early stage, thereby effectively reducing the oxidative stress of the pathological microenvironment and relieving the inflammatory response. Then, the long-term sustained release of La 3+ , so as to induce the osteogenic differentiation of MSCS and inhibit the osteoclastic activity, thereby being beneficial to the new bone formation.

[0037] Example 3: Cell compatibility detection of the sample of Example 1: (1) Cell culture: The culture of RAW264.7 cells uses high-sugar culture medium (DMEM), and the culture medium is replaced once every 1 day. The primary MSCs are isolated from the marrow cavities of the femur and tibia of the hind legs of a newborn SD rat, and are cultured using low-sugar culture medium (MEM-α).

[0038] (2) Collection of conditioned medium: first, the RAW264.7 cells are cultured on different samples for 2 days. Then, the culture medium is collected, centrifuged for 15 min, and the supernatant is mixed with the MEM-α culture medium at a volume ratio of 1:1 to form the conditioned medium of the MSC culture. Then, the osteoblasts are inoculated into different samples using the above conditioned medium.

[0039] (3) Cell compatibility detection: the RAW264.7 cells / MSCs with a cell concentration of 1×10 4 cells / cm 2 are inoculated on the surface of each sample group in a 24-well plate, 6 parallel samples are set for each group, and after the culture for 1, 3, and 5 days and 1, 3, and 7 days, respectively, the culture medium is removed and incubated for the corresponding time, then the culture medium is aspirated and each well is gently washed with PBS, then 100 μL of fresh culture medium containing MTT (0.5 mg / mL) is added to each well for further culture for 4 hours; the culture medium is aspirated, 200 μL of dimethyl sulfoxide (DMSO) solution is added to each well, then the culture plate is placed in a shaking bed for shaking for 15-20 minutes until the formazan in the well is dissolved, and finally the OD value at 490 nm is detected by an enzyme label instrument.

[0040] (4) Cell morphology observation (skeleton staining method): CLSM is used to observe the morphology of MSCs on different samples. The MSCs with a cell concentration of 1×10 4 cells / cm 2 are inoculated on the surface of each sample group in a 24-well plate, and after the culture for 3 days, the different titanium substrates are gently washed with a PBS solution, and then fixed with paraformaldehyde (4%) for 20 minutes. Rhodamine-phalloidin is used to stain the cell skeleton, and 4,6-diamidino-2-phenylindole (DAPI) is used to stain the cell nucleus. Finally, the different Ti-based materials are observed by CLSM.

[0041] (5) Cell live / dead staining experiment: RAW264.7 cells / MSCs with a concentration of 1 x 10 4 cells / cm 2 were seeded on the surface of each sample group in a 24-well plate, and three parallel samples were set for each group. After 1 day of culture, the culture solution was aspirated and the cells were washed gently with PBS. Finally, the cells were stained using a Calcein / PI cell activity and cytotoxicity detection kit, and the cells in different groups were observed by CLSM.

[0042] The results are shown in Figure 4 . Compared with other experimental groups, the MSCs on the surface of AT / La2O3 / RA were more spread and grew better. In order to detect the activity of MSCs on the surface of all samples, as shown in C of Figure 3 , the cell live / dead staining experiment showed that there was no obvious cytotoxicity of each sample group within 7 days and there were no more dead cells in each sample group. The MTT experiment also obtained similar results (as shown in D~F of Figure 3 ). Compared with other groups, the AT / La2O3 / RA group had a significant promotion of MSCs proliferation (p<0.01). In addition, as shown in A, B of Figure 5 , cell live / dead staining and MTT experiment showed that each sample group had no obvious cytotoxicity to RAW264.7 cells. The above results confirmed that each sample group had good biocompatibility.

[0043] Example 4: Detection of anti-inflammatory ability of sample of Example 1: (1) Neutral macrophages (MΦ type) play an important role in the process of responding to microenvironment inflammation. After being stimulated by the microenvironment, they can be polarized into two different functional phenotype cells, namely classical activated macrophages (M1 phenotype) and alternative activated macrophages (M2 phenotype). In order to explore the inflammatory response of macrophages on the surface of each sample group, RAW264.7 cells were treated with LPS to polarize them into inflammatory M1 type macrophages, which were used for subsequent experiments. The expression of pro-inflammatory related genes (CD86, TNF-α, MMP2) and anti-inflammatory related genes (IL-4RA, CD206, IL-10) in RAW264.7 cells on the surface of different materials was detected by RT-qPCR. As shown in C of Figure 5 , compared with the control group, AT / La2O3 / RA significantly down-regulated the expression of pro-inflammatory factors in RAW264.7 cells after 1 day of culture. The expression of pro-inflammatory related genes of AT / La(OH)3 and AT / La2O3 was significantly increased, especially in the AT / La2O3 group, due to the lower stability of the surface coating and the burst release of La ions, which is consistent with Figure 2The results were consistent with those of D. Further investigation was conducted to examine the expression of anti-inflammatory related genes in RAW264.7 cells on the surfaces of different samples, such as... Figure 5 As shown in Figure D, compared with the control group, the expression of anti-inflammatory factors in the AT, AT / La(OH)3 and AT / La2O3 groups was not significantly different from that in the control group. However, the AT / La2O3 / RA group significantly upregulated the expression of anti-inflammatory factors in RAW264.7 cells. This result is related to the effective release of RA bound to the surface of AT / La2O3 / RA.

[0044] (2) In order to further investigate the polarization of RAW264.7 cells on different sample surfaces, RAW264.7 cells induced by LPS were seeded on Ti, AT, AT / La(OH)3 and AT / La2O3, AT / La2O3 / RA surfaces. After culturing for 2 days, the fluorescence expression levels of M1 macrophage marker CD86 and M2 macrophage marker CD206 were detected by immunofluorescence staining.

[0045] Immunofluorescence staining was used to detect the expression of CD86 and CD206: After 2 days of cell culture, different titanium substrates were fixed with paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 for 10 min. After blocking with blocking buffer for 1 h, different samples were incubated overnight with rabbit monoclonal antibody. Then, after incubation with Alexa Fluor-594 secondary antibody for 1 h, cell nuclei were stained with DAPI. Finally, different samples were observed using CLSM.

[0046] The results are as follows Figure 5 The results showed that, compared with other groups, the CD86 fluorescence intensity of AT / La2O3 / RA was significantly reduced (E). p<0.01), while AT, AT / La(OH)3, and AT / La2O3 showed no significant difference compared to the Ti group. Furthermore, the green fluorescence intensity trend of CD206 on different sample surfaces was exactly the opposite of that of CD86, and the CD206 fluorescence intensity of AT / La2O3 / RA showed significant differences compared to the Ti group. (p<0.01) Similarly, AT, AT / La(OH)3, and AT / La2O3 showed no significant difference compared to the Ti group. The results of quantitative fluorescence analysis were also consistent with the above results. Figure 6 The results showed that AT / La2O3 / RA could effectively downregulate the expression of CD86 in RAW264.7 cells and upregulate the expression of CD206, promoting macrophage polarization from the M1 phenotype to the M2 phenotype.

[0047] The above results indicate that AT / La2O3 / RA can effectively inhibit macrophage inflammatory factors, promote the expression of anti-inflammatory factors, and regulate the transformation of M1 macrophages into M2 macrophages.

[0048] Example 5: In vitro osteogenic assay of different titanium-based surfaces: (1) To evaluate the ability of different titanium-based material surfaces to directly induce MSCs osteogenic differentiation, MSCs were seeded on different sample surfaces, and the expression levels of osteogenic-related genes (ALP, BMP2, OPN, and OPG) were detected, as shown in A and B of FIG. 6. After 7 days of culture, compared with the Ti group, the AT, AT / La(OH)3, AT / La2O3, and AT / La2O3 / RA groups showed a tendency of up-regulation of osteogenic-related genes, such as BMP2, OPG, and OCN, and the AT / La2O3 / RA group showed a significant difference from the other groups (p<0.01). Figure 7 In addition, it can be seen from the graph that the osteogenic ability of AT / La2O3 is lower than that of the other groups, because the cumulative release concentration of La ions is too high, which is not conducive to the osteogenic differentiation of MSCs. In addition, during the osteogenic process, the activity of alkaline phosphatase (ALP) and the mineralization ability are the markers of early and late osteogenic differentiation, respectively.

[0049] (2) To further evaluate the osteogenic induction effect of different sample coatings, ALP and mineralization staining and activity detection were performed on different samples: MSC alkaline phosphatase (ALP) activity detection: P3 MSCs were selected, seeded on the sample surface at a density of 1×10 4 cells / cm 2 and placed in a 24-well plate, and cultured under standard conditions for 3 and 7 days, with fresh medium replaced every 2 days during this period; after 3 and 7 days of culture, the culture medium was discarded, and the samples were washed with PBS buffer (5 minutes / time). 200 μL of 1% Triton X-100 was added to each well to lyse the cells, and the cells were lysed at 4°C for 30 minutes; 10 μL of the lysate was transferred to a 96-well plate, and 200 μL of BCA working solution was added. After incubation at 50°C for 20 minutes in the dark, the OD value was measured at 570 nm using a microplate reader, and the intracellular total protein concentration was calculated according to the standard curve; the ALP activity of different groups was determined according to the experimental steps of the alkaline phosphatase (ALP) activity detection kit, and the OD value of the sample at 490 nm wavelength was detected using a microplate reader; finally, the ALP activity was calculated by the formula provided in the kit (as follows) and the measured intracellular total protein amount, with the unit being U / gport. Formula: ALP activity (U / gport) = [(measured OD value-blank OD value) / (standard OD value-blank OD value)] x phenol standard concentration (0.02 mg / mL) ÷ protein concentration of sample to be tested (gport / mL).

[0050] ​Alkaline phosphatase (ALP) staining: P3 generation MSCs were selected, and staining was performed using 1×10⁻⁶ cells. 4 cells / cm 2 The samples were inoculated at a density onto the sample surface and placed in 24-well plates. They were incubated under standard conditions for 7 days, with fresh medium replaced every 2 days during this period. After 7 days of incubation, the medium was discarded, and the samples were washed with PBS buffer (5 minutes each time) and fixed with 4% paraformaldehyde. Finally, the sample surface was photographed using the BCIP / NBT alkaline phosphatase kit and an upright microscope.

[0051] MSC mineralization level detection: P3 generation MSCs were selected, at a concentration of 1×10⁻⁶ cells / cells. 4 cells / cm 2 Cells were inoculated onto the surface of each group of samples and cultured in a 37℃, 5% CO2 incubator for 7 and 14 days, with medium changes every 2 days. Mineralized nodules were observed by staining: after 3 and 7 days of culture, cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by the addition of 400 μL of 0.1% alizarin red staining solution (pH 4.1), and incubated on a shaker (80 rpm) at 37℃ for 20-30 minutes. Finally, the sample surface was washed with PBS solution before photographing using an upright microscope. After photographing, 200 μL of [unspecified substance] was added to the treated culture plate. Cells were collected after incubating in 0.2M acetic acid solution at room temperature for 30 minutes. All mixtures were transferred to new EP tubes and vortexed for 1 minute. The tubes were then placed in an 85°C water bath and heated for 10 minutes. The samples were centrifuged at low temperature (4°C, 15000 rpm, 15 minutes). 100 μL of the supernatant was aspirated into a 96-well plate and an equal volume of ammonium hydroxide (10% (v / v) 100 μL) was added. The mixture was gently vortexed and mixed. The OD values ​​of all samples were measured using a microplate reader at a wavelength of 405 nm.

[0052] The results are as follows Figure 7 As shown in Figure C, within 7 days, the AT / La2O3 / RA group exhibited the strongest ALP activity and mineralization capacity among all groups. After MSCs were continuously cultured for 14 days, the AT / La2O3 / RA group showed a stronger level of mineralization compared to other groups, clearly reflecting the strong osteoinductive ability of AT / La2O3 / RA. Quantitative data on ALP and mineralization also showed the same trend (e.g., ...). Figure 7 (D, E). Therefore, this indicates that the strong osteoinductive ability of AT / La2O3 / RA is due to the synergistic effect of La ions and RA. These results demonstrate that the AT / La2O3 / RA group has a significant advantage in inducing osteogenic differentiation of bone MSCs.

[0053] Numerous studies have confirmed that macrophages can promote osteoblast differentiation and thus new bone formation through paracrine activity. Therefore, in this invention, to investigate the effect of macrophage paracrine activity on MSCs, macrophages were seeded on different titanium-based material surfaces, cultured for one day, and the culture medium was collected as a conditioned medium for subsequent MSC culture (e.g., ...). Figure 8 (A) For example Figure 8 As shown in Figure B, different culture medium conditions did not exhibit significant cytotoxicity towards MSCs. The effects of different culture medium conditions on ALP and mineralization of MSCs inoculated on different material surfaces were then further investigated.

[0054] The results are as follows Figure 8 Figures C and D show that, compared with the Ti and AT groups, the ALP activity of MSCs in the experimental group was significantly increased after 7 days of culture. p<0.05, especially in the AT / La2O3 / RA group ( p<0.01. After MSCs were cultured for 14 days, the mineralization levels in the AT / La2O3 / RA group showed a similar trend ( Figure 8 (C, E). Further analysis of osteogenic-related gene expression levels (ALP, BMP-2, OPN, and OCN) was conducted. Figure 8 In the F and G groups, compared with the Ti group, AT-2 Mg / Ga and bone-related genes in the experimental group showed an upregulation trend. p<0.05, especially in the AT / La2O3 / RA group ( (p<0.01). The levels of osteogenic-related genes expressed by MSCs in the AT / La2O3 / RA group were higher than those under normal conditions after 7 days of culture in conditioned medium. This is because RAW264.7 cells seeded on the surface of AT / La2O3 / RA cells polarized to M2 and promoted osteogenic differentiation of MSCs through paracrine signaling.

[0055] In summary, these results indicate that AT / La2O3 / RA can not only directly induce osteogenic differentiation of MSCs, but also further promote osteogenic differentiation of MSCs through paracrine effects by inducing macrophage polarization.

[0056] Example 6: Anti-osteoclastic ability test: (1) Osteoclast induction: RAW264.7 cells were incubated at a rate of 4 × 10⁻⁶ cells / year. 4 cells / cm 2The density of RAW264.7 cells was seeded on the surface of each sample for in vitro culture. During the induction of differentiation, 50 ng / mL of receptor activator of nuclear factor kappa B ligand (RANKL) and 20 ng / mL of macrophage colony-stimulating factor (m-CSF) were added to the complete culture medium to continuously stimulate the monocyte / macrophage lineage to differentiate into multinucleated osteoclasts.

[0057] (2) TRAP detection: First, color substrate solution (dissolve color substrate in 2.5 mL detection buffer, store on ice and use within 6 hours) and standard working solution (10 μL of 10 mM p-nitrophenol diluted to 0.2 mL, final concentration 0.5 mM) were prepared; the cells in the well plate were lysed using 1% Triton X-100 lysis solution and centrifuged to obtain the supernatant, in addition, in this experiment, blank control wells, standard wells (4-40 μL gradient) and sample wells (usually 40 μL, recommended parallel or three replicate wells) should be set in a 96-well plate, mix well and incubate at 37°C for 5-30 minutes, then add 160 μL of stop solution for color development, and measure the absorbance at 405 nm (or 400-415 nm).

[0058] In this experiment, the genes related to osteoclast differentiation of RAW264.7 cells were analyzed by qRT-PCR. As shown in Figure 9 A and B, the expression levels of all osteoclast-related genes (TRAP, NFATc1, CTSK and C-FOS) in all experimental groups were significantly lower than those in the control group (p<0.05), especially in the AT / La2O3 / RA group (p<0.01). Compared with the control group, the TRAP activity of all experimental groups was significantly decreased at 3 days (p<0.05), and similarly, the AT / La2O3 / RA group was more significant (p<0.01). In addition, the fluorescence staining images of RAW264.7 cells on different material surfaces under the induction of RANKL and m-CSF factors were further observed. As shown in Figure 9 C, many differentiated multinucleated osteoclasts (white circles) appeared on Ti and AT groups, while only a small amount of osteoclasts were found on the surface of the experimental groups, especially almost no observation on the surface of AT / La2O3 / RA coating. La ions can effectively inhibit osteoclast activity. AT / La(OH)3, AT / La2O3 and AT / La2O3 / RA groups can continuously release La ions, so they can effectively inhibit the differentiation activity of osteoclasts. The AT / La2O3 / RA group performs the most outstanding in inhibiting osteoclast activity, therefore, the RA released by the AT / La2O3 / RA surface coating also has the ability to inhibit the activity of osteoclasts.​​

[0059] The above results show that La is stably released in AT / La2O3 / RA 3+ and RA, which can synergistically inhibit osteoclastogenesis, thereby facilitating the relief of bone resorption in osteoporosis.

[0060] Example 7: Evaluation of the in vivo osteogenic ability of different implants: (1) Establish an OVX SD rat model of osteoporosis to further evaluate its corresponding biological effects in pathological conditions in vivo: OVX surgical model construction: 40 SD rats were randomly divided into 5 groups and anesthetized with 2.0 wt% sodium pentobarbital / normal saline solution, and then subjected to dorsal midline depilation and iodophor disinfection, fixed in a prone position, and a longitudinal incision about 3 cm long was made on the dorsal midline, and the abdominal wall tissue was separated layer by layer and the abdominal cavity was opened. Then the fat layer was pushed aside to find the uterus, and the pink cauliflower-like ovaries at the end of the uterus were observed and separated. The oviduct was ligated, and the ovary was removed, and the contralateral ovary was removed using the same method. After 2 months, the osteoporosis model was successfully constructed, and subsequent experiments were performed.

[0061] Titanium nail implantation surgery: After 2 months of OVX model construction, a femoral defect model can be established: the rats were anesthetized with 2.0 wt% sodium pentobarbital / normal saline solution and the hind legs were disinfected and shaved, the skin and muscle were carefully cut open with a surgical blade, and the femur was exposed, a cylindrical bone defect with a depth of 1 cm and a diameter of about 1.3 mm was drilled on the surface of the femur using a surgical drill, and different groups of titanium rods were gently inserted into the bone defect until the end of the titanium rod was completely covered. The muscle and skin layers were sutured in a sterile clean bench, and the wound site was thoroughly disinfected and sterilized.

[0062] In vivo ROS level: DHE was dissolved in DMSO, and then emulsified and diluted with a mixed emulsion containing 5% Tween 80, 5% PE, glycol 400, and saline solution. 200 μL (25 μg / g) of the treated mixture was injected intravenously into each rat. After 24 hours, the SD rats were euthanized, and the femur was extracted for frozen sectioning. The prepared tissue sections were observed under a fluorescence microscope and scanned for photography.

[0063] The results are as follows: Figure 10In the middle A, by the frozen section of bone tissue and dihydroethidium (DHE, a probe that can react with ROS to emit red fluorescence) labeling, the level of ROS in vivo was observed. Compared with the pure titanium group, the ROS fluorescence intensity around the AT / La2O3 / RA group implant was significantly reduced. Therefore, it can be proved that AT / La2O3 / RA has excellent antioxidant capacity in vivo, thereby improving the microenvironment of osteoporosis excessive oxidative stress.

[0064] (2) In order to further evaluate the in vivo osteogenesis effect of different titanium-based implants, H&E and Masson tissue staining experiments were performed on bone tissue: Histological staining analysis: At 8 weeks after surgery, rats were euthanized and femur samples were collected. After fixation of the samples using paraformaldehyde, the femur samples were subjected to dialysis decalcification treatment using EDTANa2(pH 7.4) for 21 days, with decalcification solution changed every 2 days. After decalcification was completed, the titanium rod was slowly removed, and the samples were subjected to gradient concentration ethanol dehydration treatment, 15-20 minutes for each concentration. After dehydration, paraffin-embedded sections were prepared according to the experimental procedure, and the sections were subjected to H&E and Masson staining.

[0065] The results are shown in Figure 10 In the middle B, only a small amount of discontinuous osteoid tissue appeared around the pure Ti group implant. In contrast, in the experimental group, especially in the AT / La2O3 / RA implant, continuous and relatively dense new bone tissue has been formed. In addition, the Masson three-dye results also show that the collagen fibers in the new bone tissue around AT / La2O3 / RA are more abundant, indicating that the bone collagen around its implant is more mature (as shown in Figure 10 Middle C).

[0066] (3) In order to further evaluate the bone formation effect of different implant materials in vivo and the feasibility of clinical application, Micro-CT and 3D reconstruction were used to detect new bone formation around different implants. At 8 weeks after surgery, rats were euthanized and femur samples were collected; the surface skin and muscle were removed, and the samples were immersed in 4% paraformaldehyde solution for 48 hours. Viva CT400 Micro-CT was used for three-dimensional imaging scanning to evaluate the bone formation around the implant, and the analysis range was around the implant.

[0067] The results are shown in Figure 11 As shown in the middle A, compared with the Ti group, more new bone tissue can be observed on the surface of the implant in the experimental group, especially in the AT / La2O3 / RA group. In addition, as shown in Figure 11From quantitative analysis of bone volume fraction (BV / TV) and trabecular number (Tb.N), compared with Ti group, AT / La2O3 / RA group was significantly improved. While the trend of bone surface and bone volume (BS / BV) and trabecular space (Tb.Sp) was just the opposite. Therefore, AT / La2O3 / RA has a more superior ability to induce new bone formation in vivo.

[0068] Therefore, the present application constructs La2O3 / RA coating with anti-oxidation, immune regulation and bone differentiation promotion properties on the surface of titanium implant in situ by hydrothermal method and high temperature calcination technology on the surface of titanium implant, i.e. AT / La2O3 / RA. Experimental results show that AT / La2O3 / RA can continuously and stably release RA and La 3+ In the simulated osteoporosis pathological microenvironment, AT / La2O3 / RA can effectively scavenge ROS, and significantly promote the polarization of macrophages to M2, and up-regulate the expression level of anti-inflammatory factors and down-regulate the expression level of pro-inflammatory factors, so as to alleviate the inflammatory response. In addition, AT / La2O3 / RA also has the ability to significantly promote the osteogenic differentiation of BMSCs and inhibit the differentiation of osteoclasts. In the OVX osteoporosis SD rat model, AT / La2O3 / RA has a significant anti-oxidative stress effect and inhibits inflammatory response in vivo, thereby effectively promoting bone integration in vivo.

[0069] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application but not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A multifunctional composite coating for titanium implants, characterized in that, The coating is formed in situ on the surface of a titanium or titanium alloy implant, and comprises a La2O3 nanoparticle layer and a radix paeoniae alba glycoside A loaded on the La2O3 nanoparticle layer.

2. A method for producing a multifunctional composite coating of a titanium implant according to claim 1, characterized in that, The method comprises the following steps: S1, subjecting a titanium or titanium alloy substrate to an alkali heat treatment to obtain a titanium implant surface AT; S2, in-situ synthesizing a La(OH)3 coating on the AT through a hydrothermal reaction to obtain AT / La(OH)3; S3, calcining the AT / La(OH)3 to convert the La(OH)3 into La2O3 to obtain AT / La2O3; S4, immersing the AT / La2O3 in a radix paeoniae alba glycoside A solution to load the RA into the porous structure of the coating to obtain an AT / La2O3 / RA composite coating.

3. The preparation method according to claim 2, characterized in that, In S1, the alkali heat treatment is to ultrasonically wash a pure titanium containing a titanium disc and a titanium rod with ethanol and deionized water for 1 hour, and then react the pure titanium in a 4-6 M NaOH solution at 70-90°C for 24 hours.

4. The production method according to claim 2, characterized by, In S2, the hydrothermal reaction is to heat a reaction kettle containing 10 mL of a hydrothermal reaction solution composed of urea and La(NO3)3·6H2O in a molar ratio of 1:1 at 100-120°C for 12 hours.

5. The preparation method according to claim 2, characterized in that, In S3, the calcination temperature is 600-800°C, and the time is 2 hours.

6. The preparation method according to claim 2, characterized in that, In S4, the concentration of the radix paeoniae alba glycoside A solution is 50-200 μg / mL.

7. Use of the titanium implant multifunctional composite coating according to claim 1 in the preparation of an implant material for repairing osteoporotic bone defects.

8. Use according to claim 7, characterized in that, The titanium implant multifunctional composite coating sustained release La 3 + and wogonoside A.

9. Use according to claim 7, characterized in that, The titanium implant multifunctional composite coating has antioxidant activity and anti-inflammatory effect, promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and inhibits the differentiation of osteoclasts.

10. Use according to claim 7, characterized in that, It is used for repairing bone defects of osteoporosis patients and promoting new bone formation and osseointegration.

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