A multifunctional composite coating for titanium implants with dual properties of resisting reactive oxygen species and anti-inflammation, and its preparation method.

Abstract

This application relates to the field of biomedical materials technology, specifically to a multifunctional composite coating for titanium implants with dual properties of resisting reactive oxygen species and anti-inflammation, and its preparation method. The coating consists of a layer of lanthanum oxide (La2O3) nanoparticles grown in situ on the surface of the titanium implant and lanthanum glycoside A (RA) loaded thereon, capable of continuously releasing La2O3. 3+ The preparation method includes: alkaline heat treatment of a titanium substrate; in-situ synthesis of a La(OH)3 coating on the surface via hydrothermal reaction; calcination to transform it into a porous La2O3 nanoparticle layer; and finally, loading RA via impregnation. This coating can effectively scavenge ROS, regulate immune inflammatory responses, promote osteogenic differentiation, and inhibit osteoclast activity, thereby significantly promoting bone integration. This invention effectively improves the challenge of bone integration in the pathological microenvironment of osteoporosis. The preparation process is simple and controllable, suitable for bone defect repair in patients with osteoporosis, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a multifunctional composite coating for titanium implants with dual properties of resisting reactive oxygen species and anti-inflammation, and its preparation method. Background Technology

[0002] Osteoporosis, a systemic metabolic disease of the bones, is characterized at the macroscopic level by decreased bone mass, deterioration of bone microstructure, and reduced mechanical properties, leading to a significantly increased risk of fractures. At the microscopic level, the disease exhibits multidimensional characteristics: at the microenvironment level, there is a decrease in pH, an abnormally high level of reactive oxygen species (ROS), and a chronic inflammatory state; at the cellular metabolic level, there is a decline in the osteogenic differentiation capacity of mesenchymal stem cells, an increase in osteoclast activity, and an imbalance in macrophage polarization (with M1 type dominating). This multi-level homeostasis imbalance leads to a prolonged repair cycle of osteoporotic bone defects, exceeding 3 months, far exceeding the healing rate of normal bone tissue (usually <2 months).

[0003] Titanium and titanium alloys, with their excellent mechanical strength, corrosion resistance, and biocompatibility, have become the mainstream implant materials for repairing osteoporotic bone defects. However, their inherent bioinert surface properties severely limit the integration efficiency between the implant and the host bone tissue. In the pathological microenvironment of osteoporosis, abnormally increased osteoclast activity and suppressed osteogenic capacity create a vicious cycle, hindering the formation of the peri-implant bone integration interface and significantly increasing the incidence of early postoperative loosening and displacement. More seriously, oxidative stress and chronic inflammation in the pathological microenvironment further inhibit new bone formation by disrupting intercellular networks (including cytokine signaling, material-cell interface interactions, and intercellular interactions). Clinical data show that the failure rate of bone implantation surgery in osteoporotic patients is approximately 40% higher than in healthy individuals, highlighting the urgent need to improve bone integration performance under pathological conditions.

[0004] It is noteworthy that recent studies have found that regulating microenvironmental homeostasis (such as inhibiting oxidative stress and modulating inflammation levels) can effectively reverse the progression of osteoporosis and promote bone regeneration. However, these strategies are often single-function and cannot synergistically address the multiple obstacles coexisting in the osteoporotic microenvironment, such as oxidative stress, inflammation, osteogenic inhibition, and osteoclastosis.

[0005] In recent years, rare earth oxides such as lanthanum oxide (La2O3) have attracted attention as ideal nanocarriers due to their excellent biocompatibility, controllable ion release, and abundant mesoporous structure, and their potential osteogenic induction and osteoclast inhibition capabilities. Meanwhile, active ingredients extracted from natural plants, such as Regaloside A (RA), have been shown to possess excellent antioxidant and anti-inflammatory properties. However, no technology currently exists to synergistically integrate these two properties onto the surface of titanium implants to construct a multifunctional composite coating capable of simultaneously addressing multiple challenges in the osteoporotic pathological microenvironment, including oxidative stress, chronic inflammation, osteogenic inhibition, and osteoclastosis. Therefore, developing a titanium implant composite coating that integrates the advantages of both is crucial for significantly improving bone repair efficacy and long-term implant stability under osteoporotic pathological conditions. Summary of the Invention

[0006] The purpose of this invention is to provide a multifunctional composite coating for titanium implants with dual properties of resisting reactive oxygen species and anti-inflammation, and a method for preparing the same. This coating can effectively remove ROS, regulate immune inflammatory responses, promote osteogenic differentiation and inhibit osteoclast activity under osteoporotic pathological conditions, thereby significantly promoting osseointegration.

[0007] To achieve the above objectives, on the one hand, the present invention provides a multifunctional composite coating for titanium implants, wherein the coating is formed in situ on the surface of a titanium or titanium alloy implant, and includes a La2O3 nanoparticle layer and a lily glycoside A loaded on the La2O3 nanoparticle layer.

[0008] On the other hand, the present invention also provides a method for preparing a multifunctional composite coating for titanium implants, comprising the following steps:

[0009] S1. Alkali heat treatment is performed on the titanium or titanium alloy substrate to obtain the AT surface of the titanium implant.

[0010] S2. The AT is synthesized in situ with a La(OH)3 coating by hydrothermal reaction to obtain AT / La(OH)3;

[0011] S3. Calcine the AT / La(OH)3 to convert La(OH)3 into La2O3 to obtain AT / La2O3;

[0012] S4. Immerse the AT / La2O3 in a solution of lily glycoside A to load RA into the porous structure of the coating, thereby obtaining an AT / La2O3 / RA composite coating.

[0013] Further, in S1, the alkaline heat treatment involves ultrasonically washing the titanium disc and titanium rod with ethanol and deionized water for 1 hour each, and then reacting the pure titanium with the titanium disc and titanium rod in a 4-6 M NaOH solution at 70-90 °C for 24 hours.

[0014] Further, in S2, the hydrothermal reaction involves placing AT in a reactor containing a 10 mL hydrothermal reaction solution of urea and La(NO3)3·6H2O in a molar ratio of 1:1 and heating it at 100~120°C for 12 hours.

[0015] Furthermore, in S3, the calcination temperature is 600~800℃ and the time is 2 hours.

[0016] Furthermore, in S4, the concentration of the lily glycoside A solution is 50~200 μg / mL.

[0017] The present invention also provides the application of a multifunctional composite coating for titanium implants in the preparation of implant materials for the repair of osteoporotic bone defects.

[0018] Furthermore, the multifunctional composite coating of the titanium implant continuously releases La 3+ And Wang Baihe Glycoside A.

[0019] Furthermore, the multifunctional composite coating of the titanium implant has antioxidant and anti-inflammatory effects; it promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) and inhibits osteoclast differentiation.

[0020] Furthermore, it can be used to repair bone defects in patients with osteoporosis, promoting new bone formation and bone integration.

[0021] The beneficial effects of the multifunctional composite coating for titanium implants with dual properties of resisting reactive oxygen species and anti-inflammation, as described in this invention, and its preparation method, are as follows:

[0022] (1) Multifunctional synergistic effect: This invention is the first to combine La2O3, which has osteogenic / osteoclastogenic regulation capabilities, with RA, which has strong antioxidant / anti-inflammatory capabilities, to construct a multifunctional coating. La 3+ Synergistic release with RA can simultaneously intervene in multiple key factors of the osteoporosis pathological microenvironment (high ROS, chronic inflammation, osteogenic inhibition).

[0023] (2) Excellent antioxidant properties: The loaded RA can effectively remove excess ROS in the pathological microenvironment, alleviate oxidative stress, and create favorable conditions for bone regeneration.

[0024] (3) Effective immunomodulation and anti-inflammatory effects: The coating can promote the polarization of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type, upregulate anti-inflammatory factors and downregulate pro-inflammatory factors, thereby reducing the inflammatory response around the implant.

[0025] (4) Powerful ability to promote bone formation and inhibit bone resorption: Continuously released La 3+ It can directly promote osteogenic differentiation of MSCs and upregulate the expression of osteogenic-related genes; at the same time, La 3+Both RA and RA can effectively inhibit osteoclast differentiation and activity, and downregulate the expression of osteoclast-related genes.

[0026] (5) Improved osseointegration: In animal models of osteoporosis, the coating of the present invention significantly reduced the ROS level and inflammatory response around the implant, promoted the formation of a large amount of mature new bone, increased the bone volume fraction and the number of trabeculae, and demonstrated excellent in vivo osseointegration ability.

[0027] (6) The preparation process is simple and controllable: The present invention uses conventional methods such as hydrothermal treatment, calcination, and impregnation. The process is simple, has good repeatability, and is easy to achieve large-scale production.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0029] Figure 1 A schematic diagram illustrating the design strategy of AT / La2O3 / RA in scavenging reactive oxygen species (ROS), regulating immune responses, and highlighting its potential to promote osteointegration in the osteoporotic microenvironment;

[0030] Figure 2 Image A shows scanning electron microscope (SEM) and energy-dispersive spectroscopy (EDS) images of all samples (white scale bars are 10 μm and 50 μm, respectively); image B shows X-ray diffraction (XRD) analysis of AT / La(OH)3 and AT / La2O3; image C shows the water contact angle of different samples; and image D shows the La content in AT / La(OH)3, AT / La2O3, and AT / La2O3 / RA. 3+ The cumulative release amount, E is the cumulative release amount of RA in AT / La2O3 / RA;

[0031] Figure 3 In the table, A represents the H2O2 scavenging capacity of all coated samples, B represents the total antioxidant capacity analysis of all samples, C represents the cell viability and death staining and cytoskeleton staining results of MSCs after 7 days of treatment with different samples (scale bar: 20 μm), D represents the quantitative analysis of MSC proliferation at 1 day under different sample treatments, E represents the quantitative analysis of MSC proliferation at 3 days under different sample treatments, and F represents the quantitative analysis of MSC proliferation at 7 days under different sample treatments.

[0032] Figure 4 The cytoskeleton staining diagram of MSCs from different samples;

[0033] 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;

[0034] Figure 6 This is a quantitative immunofluorescence analysis of CD86 and CD206.

[0035] 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.

[0036] 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;

[0037] 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.

[0038] 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.

[0039] Figure 11 Image A shows the assessment of new bone formation using micro-CT scan two months after surgical implantation; image B shows the quantitative statistical results of bone volume fraction (BV / TV); image C shows the quantitative statistical results of trabecular meshwork (Tb.N); and image D shows the quantitative statistical results of intertrabecular spacing (Tb.Sp). p<0.01 . Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0042] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0043] This invention designs a coating of lanthanum oxide nanoparticles loaded with RA (e.g., based on the characteristics of the pathological microenvironment of osteoporosis) Figure 1 (As shown).

[0044] Example 1:

[0045] Creating a composite layer:

[0046] After ultrasonic washing with ethanol and deionized water for 1 h each, pure titanium containing titanium discs and rods was reacted in 5 M NaOH solution at 80 °C for 1 day. The treated sample was named AT (alkaline heat treatment). The AT sample was placed in a reactor with a 10 mL hydrothermal reaction solution composed of 21.6 mM urea and La(NO3)3·6H2O and heated at 105 °C for 12 h. The treated sample was named AT / La(OH)3. The obtained sample was then calcined in a muffle furnace at 750 °C for 2 h. The resulting sample was named AT / La2O3. Finally, the calcined sample was soaked in a 100 μg / mL solution of lily glycosides overnight to obtain the final sample, which was named AT / La2O3 / RA.

[0047] Example 2:

[0048] Characterization of the sample in Example 1:

[0049] (1) The surface morphology of all samples was characterized by SEM. For example... Figure 2 As shown in Figure A, the pure Ti sample exhibits a relatively smooth surface morphology, while other groups show relatively rough nanostructures. At higher magnifications, AT reveals numerous nanoscale cross grooves. Smaller needle-like and spindle-shaped nanoparticle structures can be observed on the AT / La(OH)3 coating surface, while the AT / La2O3 surface shows more larger spindle-shaped nanoparticle structures. When La(OH)3 is calcined to La2O3, the intergranular interface energy increases with increasing temperature, which is more favorable for grain boundary migration, resulting in faster grain growth and larger grain size. Furthermore, the pore size of the particles increases when metal hydroxides transform into oxides; therefore, the porous internal structure of the particles after the transformation of La(OH)3 to La2O3 is more conducive to loading RA. Figure 2 As shown in Figure A, compared to the AT / La2O3 coating, the nanostructure on the surface of the AT / La2O3 / RA coating becomes blurred. This is because the La2O3 nanoparticles loaded with RA form a film-like structure on the coating surface, thus proving that lycopene has been successfully encapsulated. Furthermore, EDS characterization revealed the distribution and composition of elements on the material surface. Figure 2 As shown in Figure A, compared with the control group, the AT / La(OH)3, AT / La2O3 and AT / La2O3 / RA groups showed a large amount of La with a content of over 50%. In addition, the AT / La2O3 / RA group showed a large amount of C element with a content of 27%, which mainly came from RA. This also proves that RA has been successfully loaded into the coating.

[0050] (2) The crystal phases of various samples were analyzed by XRD, such as Figure 2 As shown in Figure B, samples AT / La(OH)3 and AT / La2O3 both exhibit characteristic peaks of the corresponding substances, indicating that the present invention successfully synthesized La(OH)3 and AT / La2O3 in situ on the surface of AT / La(OH)3 and AT / La2O3 samples.

[0051] (3) The hydrophilicity or hydrophobicity of the surface of biomaterials is also one of the key factors regulating cell behavior. For example, Figure 2 As shown in Figure C, the hydrophilicity and hydrophobicity of each sample surface were detected using a water contact angle meter. The results showed that the water contact angles of Ti, AT, AT / La(OH)3, AT / La2O3, and AT / La2O3 / 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 coatings in the experimental group was significantly smaller than that of the pure titanium surface, indicating that the hydrophilicity of the titanium surface was greatly improved after functionalization modification, which will be beneficial to the adhesion of MSCs to the material surface.

[0052] (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 different time intervals (0, 1, 3, 7, 11, 14 and 21 days) at 37°C; finally, the collected liquid was centrifuged at high speed (4°C, 12000 rpm, 10 minutes), 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.

[0053] (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.

[0054] In summary, the above demonstrates that the AT / La2O3 / RA coating preferentially releases RA in the early stages, effectively reducing oxidative stress in the pathological microenvironment and alleviating the inflammatory response. Subsequently, the long-term continuous release of La... 3+ The aim is to induce osteogenic differentiation of MSCS and inhibit osteoclast activity, thereby promoting new bone formation.

[0055] Example 3:

[0056] Example 1: Cell compatibility testing of samples:

[0057] (1) Cell culture: RAW264.7 cells were cultured in high glucose medium (DMEM) and the medium was changed every day. Primary MSCs were isolated from the femur and tibial medullary cavity of newborn SD rats and cultured in low glucose medium (MEM-α).

[0058] (2) Collection of conditioned medium: First, RAW264.7 cells were cultured on different samples for 2 days. Then, the medium was collected, centrifuged for 15 min, and the supernatant was mixed with MEM-α medium at a volume ratio of 1:1 to form the conditioned medium for MSC culture. Then, osteoblasts were seeded into different samples using the above conditioned medium.

[0059] (3) Cell compatibility test: Cells were placed in 24-well plates at a concentration of 1×10⁶ cells / well. 4 cells / cm 2 RAW264.7 cells / MSCs were seeded onto the surface of each sample group, with 6 replicates per group. After culturing for 1, 3, and 5 days, and 1, 3, and 7 days respectively, the culture medium was removed and incubated for the corresponding time. The culture medium was then aspirated, and each well was gently rinsed with PBS. 100 μL of fresh culture medium containing MTT (0.5 mg / mL) was added to each well, and the cells were cultured for another 4 hours. The culture medium was then aspirated, and 200 μL of dimethyl sulfoxide (DMSO) solution was added to each well. The culture plate was then placed in a shaker and shaken for 15-20 minutes until the formazan in the wells dissolved. Finally, the OD value at 490 nm was measured using a microplate reader.

[0060] (4) Cell morphology observation (scaffold staining method): CLSM was used to observe the morphology of MSCs on different samples. Cells were placed in 24-well plates at a concentration of 1×10⁶ cells / well. 4 cells / cm 2 MSCs were seeded onto the surfaces of different sample groups and cultured for 3 days. Different titanium substrates were gently washed with PBS solution and then fixed with paraformaldehyde (4%) for 20 minutes. The cytoskeleton was stained with rhodamine-phalloidin, and the cell nuclei were stained with 4,6-diamidin-2-phenylindole (DAPI). Finally, the different Ti-based materials were observed using CLSM.

[0061] (5) Cell liveness / death staining experiment: In a 24-well plate, the cell concentration was 1×10⁶ cells / wells. 4 cells / cm 2 RAW264.7 cells / MSCs were seeded on the surface of each sample group, with 3 parallel samples in each group. After culturing for 1 day, the culture medium was aspirated and the cells were gently washed once with PBS. Finally, the cells were stained with Calcein / PI cell viability and cytotoxicity assay kit, and the cells in different groups were observed by CLSM.

[0062] The results are as follows Figure 4 As shown, compared with other experimental groups, MSCs on the AT / La2O3 / RA surface exhibited a more spread morphology and better growth. To detect the activity of MSCs on the surfaces of all samples, as shown... Figure 3 As shown in Figure C, the cell viability staining results indicated that none of the samples exhibited significant cytotoxicity within 7 days, and none of the samples showed a large number of dead cells. The MTT assay also yielded similar results (e.g., ...). Figure 3 (D~F). Compared with other groups, the AT / La2O3 / RA group significantly promoted MSC proliferation (p<0.01). Additionally, as... Figure 5 As shown in Figures A and B, cell viability staining and MTT assays revealed that none of the samples exhibited significant cytotoxicity against RAW264.7 cells. These results confirm that all samples possess good biocompatibility.

[0063] Example 4:

[0064] Example 1: Anti-inflammatory ability test of the sample:

[0065] (1) Neutral macrophages (MΦ type), which play an important role in the response to inflammatory reactions in the microenvironment, can polarize into two different functional phenotypes after stimulation by the microenvironment: classical activated macrophages (M1 phenotype) and alternative activated macrophages (M2 phenotype). To investigate the inflammatory response of macrophages on the surfaces of various samples, RAW264.7 cells were treated with LPS to polarize them into inflammatory M1 type macrophages, which were then used for subsequent experiments. The expression of pro-inflammatory genes (CD86, TNF-α, MMP2) and anti-inflammatory genes (IL-4RA, CD206, IL-10) in RAW264.7 cells on different material surfaces was detected by RT-qPCR. Figure 5 As shown in Figure C, after one day of culture, AT / La2O3 / RA significantly downregulated the expression of pro-inflammatory factors in RAW264.7 cells compared to the control group. The expression of pro-inflammatory genes in AT / La(OH)3 and AT / La2O3 was significantly increased, especially in the AT / La2O3 group. This is attributed to the lower stability of its surface coating and the resulting La ion release, which is related to… Figure 2 The 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.

[0066] (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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Example 5:

[0071] In vitro osteogenic assays of different titanium-based surfaces:

[0072] (1) To evaluate the ability of different titanium-based materials to directly induce osteogenic differentiation of MSCs, MSCs were seeded on the surfaces of different samples, and the expression levels of osteogenic-related genes (ALP, BMP2, OPN, and OPG) were detected, such as Figure 7 As shown in Figures A and B, after 7 days of culture, compared with the Ti group, AT, AT / La(OH)3, AT / La2O3, and AT / La2O3 / RA, which constitute bone-related genes such as BMP2, OPG, and OCN, showed an upregulation trend. In particular, the AT / La2O3 / RA group showed significant differences from other groups. (p<0.01). Furthermore, the graph shows that the osteogenic capacity of AT / La2O3 was lower than other groups because the cumulative release concentration of La ions was too high, which is unfavorable for osteogenic differentiation of MSCs. Additionally, during osteogenic processes, alkaline phosphatase (ALP) activity and mineralization capacity are markers of early and late osteogenic differentiation, respectively.

[0073] (2) To further evaluate the osteogenic induction effect of different sample coatings, ALP and mineralization staining and activity detection were performed on different samples:

[0074] MSCs alkaline phosphatase (ALP) activity assay: P3 generation MSCs were selected, and 1×10⁻⁶ cells were used to detect ALP activity. 4 cells / cm 2Cells were seeded at high density onto the sample surface and placed in 24-well plates. They were cultured under standard conditions for 3 and 7 days, with fresh medium replaced every 2 days. After 3 and 7 days, the medium was discarded, and the samples were washed with PBS buffer (5 minutes each time). 200 μL of 1% Triton X-100 was added to each well to lyse the cells at 4°C for 30 minutes. 10 μL of the lysis buffer was transferred to a 96-well plate, and 200 μL of BCA working solution was added. After incubation at 50°C in the dark for 20 minutes, the OD value was measured at 570 nm using a microplate reader, and the intracellular total protein concentration was calculated based on the standard curve. ALP activity in different groups was measured according to the experimental steps of the alkaline phosphatase (ALP) activity assay kit, and the OD value of the samples was measured at 490 nm using a microplate reader. Finally, ALP activity was calculated using the formula provided in the kit (as shown below) and the measured intracellular total protein amount, with units of U / gport. Formula: ALP activity (U / gport) = [(Measured OD value - Blank OD value) / (Standard OD value - Blank OD value)] × Phenol standard concentration (0.02 mg / mL) ÷ Protein concentration of the sample to be tested (gport / mL).

[0075] 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.

[0076] MSC mineralization level detection: P3 generation MSCs were selected, at a concentration of 1×10⁻⁶ cells / cells. 4 cells / cm 2Cells 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] Example 6:

[0082] Anti-osteoclastic ability test:

[0083] (1) Osteoclast induction: RAW264.7 cells were incubated at a rate of 4 × 10⁻⁶ cells / year. 4 cells / cm 2 The cells were seeded at different densities onto the surface of each sample for in vitro culture. During the differentiation induction process, 50 ng / mL of receptor activator of nuclear factor κB ligand (RANKL) and 20 ng / mL of macrophage colony-stimulating factor (m-CSF) were added to the complete culture medium to guide the monocyte / macrophage cell line toward multinucleated osteoclasts through continuous stimulation.

[0084] (2) TRAP detection: First, prepare the chromogenic substrate solution (dissolve the chromogenic substrate in 2.5 mL of detection buffer, store on ice and use within 6 hours) and the standard working solution (10 μL of 10 mM p-nitrophenol diluted to 0.2 mL, final concentration 0.5 mM); lyse the cells in the well plate with 1% Triton X-100 lysis buffer and centrifuge to collect the supernatant. In addition, in this experiment, a 96-well plate should be used to set up blank control wells, standard wells (4-40 μL gradient) and sample wells (usually 40 μL, parallel or triplet wells are recommended). After mixing, incubate at 37℃ for 5-30 minutes, then add 160 μL of stop solution to each well for color development, and measure the absorbance at 405 nm (or 400-415 nm).

[0085] In this experiment, genes related to osteoclast differentiation in RAW264.7 cells were analyzed by qRT-PCR. For example... Figure 9 As shown in Figures A and B, the expression levels of 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. Meanwhile, compared with the control group, TRAP activity in all experimental groups decreased significantly at 3 days (p<0.01). p<0.05), and similarly, the AT / La2O3 / RA group showed a more significant difference (p<0.05). (p<0.01). Furthermore, fluorescence staining images of RAW264.7 cells on different material surfaces induced by RANKL and m-CSF factors were observed. Figure 9 As shown in Figure C, numerous differentiated multinucleated osteoclasts (white circles) appeared on the Ti and AT groups, while only a small number of osteoclasts were observed on the surface of the experimental groups, especially on the AT / La2O3 / RA coating surface, where almost none were observed. La ions can effectively inhibit osteoclast activity. The surface coatings of AT / La(OH)3, AT / La2O3, and AT / La2O3 / RA groups can continuously release La ions, thus effectively inhibiting osteoclast differentiation activity. The AT / La2O3 / RA group showed the most prominent effect in inhibiting osteoclast activity; therefore, the RA released by the AT / La2O3 / RA surface coating also has the ability to inhibit osteoclast activity.

[0086] The results in summary indicate that La is stably released from AT / La2O3 / RA. 3+ Together with RA, they can synergistically inhibit osteoclast formation, thereby helping to alleviate bone resorption in osteoporosis.

[0087] Example 7:

[0088] Assessment of osteogenesis capacity of different implants:

[0089] (1) Establish an osteoporosis model in OVX-type SD rats and further evaluate its corresponding biological effects in vivo under pathological conditions:

[0090] OVX surgical model construction: Forty SD rats were randomly divided into 5 groups and anesthetized with 2.0wt% sodium pentobarbital / physiological saline solution. The rats were then shaved and disinfected with iodine along the midline of their backs. The rats were fixed in a prone position, and a longitudinal incision of about 3 cm was made along the midline of their backs. The abdominal wall tissues were separated layer by layer and the abdominal cavity was opened. The uterus was then located by separating the fat layer. The pink cauliflower-shaped ovary could be observed and separated at the end of the uterus. The fallopian tubes were ligated, the ovary was removed, and the contralateral ovary was removed using the same method. Two months later, the osteoporosis model was successfully established, and subsequent experiments were conducted.

[0091] Titanium nail implantation surgery: Two months after the OVX model was established, a femoral defect model could be created: Rats were anesthetized with 2.0wt% sodium pentobarbital / physiological saline solution, and the hind legs were disinfected and shaved. The skin and muscles were carefully cut with a scalpel 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 femoral surface with a surgical drill. Different groups of titanium rods were gently inserted into the bone defect until the end of the titanium rod completely covered the defect. The wound was sutured to the muscle and skin layers in a sterile laminar flow hood, and the wound site was thoroughly disinfected and sterilized.

[0092] In vivo ROS levels: DHE was dissolved in DMSO and then emulsified and diluted with a mixed emulsion containing 5% Tween 80, 5% PE, ethylene glycol 400 and saline solution. 200 μL (25 μg / g) of the prepared mixture was injected intravenously into each rat. SD rats were euthanized 24 hours later, and the femurs of the rats were extracted and frozen sections were prepared. The prepared tissue sections were observed under a fluorescence microscope and scanned and photographed.

[0093] The results are as follows Figure 10 As shown in Figure A, the level of ROS in vivo was observed by labeling frozen sections of bone tissue with dihydroethidium (DHE, a probe that emits red fluorescence after reacting with ROS). Compared with the pure titanium group, the AT / La2O3 / RA group showed a significant decrease in ROS fluorescence intensity around the implant. Therefore, it can be demonstrated that AT / La2O3 / RA has excellent antioxidant capacity in vivo, thereby improving the microenvironment of osteoporosis-induced excessive oxidative stress.

[0094] (2) To further evaluate the in vivo osteogenic effect of different titanium-based implants, H&E and Masson staining experiments were performed on bone tissue:

[0095] Histological staining analysis: Eight weeks after surgery, rats were euthanized and femoral samples were collected. After fixation with paraformaldehyde, the femoral samples were dialyzed and decalcified with EDTANa2 (pH 7.4) for 21 days, with the decalcification solution changed every 2 days. After decalcification was completed, the titanium rod was slowly removed, and the samples were sequentially dehydrated with ethanol of varying concentrations for 15-20 minutes at each concentration. After dehydration, paraffin-embedded sections were prepared according to the experimental procedure, and the sections were stained with H&E and Masson staining.

[0096] The results are as follows Figure 10 As shown in Figure B, only a small amount of discontinuous osteoid tissue appeared around the implant in the pure Ti group. In contrast, the experimental groups, especially the AT / La2O3 / RA implant, had formed continuous and relatively dense new bone tissue. Furthermore, Masson triple staining results also showed richer collagen fiber expression in the new bone tissue around the AT / La2O3 / RA implant, indicating that the bone collagen around the implant was more mature (e.g., ...). Figure 10 (C)

[0097] (3) 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. Eight weeks after surgery, rats were euthanized and femoral samples were collected; the surface skin and muscle were removed, and the samples were soaked in 4% paraformaldehyde solution for 48 hours. Three-dimensional imaging scans were performed on the samples using Viva CT400 Micro-CT to evaluate bone formation around the implants. The analysis range was around the implants.

[0098] The results are as follows Figure 11 As shown in Figure A, compared to the Ti group, more new bone tissue was observed on the implant surface in the experimental groups, especially in the AT / La2O3 / RA group. Furthermore, as... Figure 11 As shown in Figures B-D, quantitative analysis of bone volume fraction (BV / TV) and trabecular meshwork (Tb.N) showed significant increases in the AT / La2O3 / RA group compared to the Ti group. However, the trends for bone surface area and bone volume (BS / BV) and trabecular meshwork (Tb.Sp) were exactly opposite. Therefore, the AT / La2O3 / RA group exhibits a superior ability to induce new bone formation in vivo.

[0099] Therefore, this invention constructs an in-situ La2O3 / RA coating, namely AT / La2O3 / RA, on the surface of titanium implants using hydrothermal methods and high-temperature calcination. The coating possesses antioxidant, immunomodulatory, and osteodifferentiation-promoting properties. Experimental results show that AT / La2O3 / RA can continuously and stably release RA and La. 3+In a simulated osteoporotic microenvironment, AT / La2O3 / RA effectively scavenged ROS, significantly promoted macrophage M2 polarization, and upregulated and downregulated the expression levels of anti-inflammatory and pro-inflammatory factors, thereby alleviating the inflammatory response. Furthermore, AT / La2O3 / RA also significantly promoted osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and inhibited osteoclast differentiation. In the OVX osteoporosis SD rat model, AT / La2O3 / RA exhibited significant antioxidant stress and anti-inflammatory effects in vivo, effectively promoting bone integration.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

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 method of claim 2, wherein, 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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