A light-heat-electricity coupling titanium-based implant and a preparation method and application thereof

Abstract

The application discloses a light-heat-electricity coupling titanium-based implant as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The light-heat-electricity coupling titanium-based implant provided by the application comprises a porous titanium substrate and a hydrogel layer; the surface of the porous titanium substrate is a TiO2 nanotube array structure; up-conversion nanoparticles and g-C3N4 nanoparticles are arranged between the porous titanium substrate and the hydrogel layer, the up-conversion nanoparticles are Yb 3+ and Er 3+ co-doped NaYF4 up-conversion nanoparticles; and the hydrogel layer is loaded with carbon nanotubes and a medicinal active ingredient. The application realizes the synergic coupling of light-heat-electricity multi-energy forms, endows the titanium-based implant with the integrated functions of light response, heat conversion, electric signal conduction and local treatment, and provides an integrated solution with structural support, biological activity regulation and treatment for bone tissue defect repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a light-heat-electricity coupling titanium-based implant as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those of ordinary skill in the art.

[0003] Osteosarcoma is a common primary malignant tumor in children and adolescents. Current clinical treatment mainly combines tumor resection with chemotherapy and radiotherapy as auxiliary means, aiming to eliminate residual tumor cells to reduce the risk of recurrence. However, such traditional treatment has significant limitations: chemotherapy and radiotherapy, while acting on tumor cells, often cause non-specific damage to surrounding normal tissues and cells, affecting patient recovery and quality of life after surgery; in addition, even after comprehensive treatment, there may still be tumor tissue that has not been completely eliminated, and these residual lesions pose a potential risk of tumor recurrence. During the repair of bone tissue defects after tumor resection, the filling site is prone to bacterial infection due to surgical trauma and changes in local immune status, further exacerbating the treatment difficulty. At the same time, there are significant differences in the shape, size, and anatomical structure of bone defects among individual patients, making it difficult for traditional titanium implants to achieve personalized adaptation and efficient mass production, limiting their widespread application in clinical settings.

[0004] In the field of bone tissue defect repair materials, 3D-printed titanium and its alloys have gradually become the mainstream choice and are widely used due to their good mechanical properties, biocompatibility, and flexibility in structural design. However, existing 3D-printed titanium implants still have functional shortcomings: their surface has limited biological bonding capacity with surrounding bone tissue, making it difficult to achieve rapid and stable bone integration; at the same time, they lack effective active intervention capabilities in inhibiting tumor recurrence and preventing infection, failing to meet the clinical demand for multi-target synergistic therapy in bone repair, anti-tumor, and anti-infection, which collectively restricts the further improvement of their therapeutic effect. SUMMARY

[0005] Therefore, the present application provides a light-heat-electricity coupling titanium-based implant as well as a preparation method and application thereof. The present application constructs a light-heat-electricity coupling functional coating layer integrating upconversion nanoparticles, g-C3N4 / TiO2 heterojunction, and hydrogel layer on the surface of a porous titanium substrate, which can synergistically achieve efficient bone bonding, antibacterial, and antitumor effects under near-infrared light excitation.

[0006] In a first aspect, the present application provides a light-heat-electricity coupling titanium-based implant, comprising a porous titanium substrate and a hydrogel layer.

[0007] The surface of the porous titanium substrate is a TiO2 nanotube array structure; the porous titanium substrate and the hydrogel layer are provided with upconversion nanoparticles and g-C3N4 nanoparticles, the upconversion nanoparticles are Yb 3+ and Er 3+ co-doped NaYF4 upconversion nanoparticles; the hydrogel layer is loaded with carbon nanotubes and a pharmaceutically active ingredient.

[0008] Preferably, the material of the hydrogel layer is a gelatin-sodium alginate hydrogel; the pharmaceutically active ingredient includes curcumin; in the hydrogel layer, the use amount ratio of carbon nanotubes, the pharmaceutically active ingredient, gelatin and sodium alginate is (0.05~0.15) μg : (20~50) μg : (0.08~0.12) g : (0.01~0.04) g.

[0009] Preferably, in the upconversion nanoparticles, the doping molar concentration of Yb 3+ is 15~25%, and the doping molar concentration of Er 3+ is 1~5%.

[0010] Preferably, the porous titanium substrate is pure titanium or a titanium alloy, and the porosity of the porous titanium substrate is 50~90%.

[0011] In a second aspect, the application provides a preparation method of the above-mentioned light-heat-electricity coupling titanium-based implant, comprising the following steps:

[0012] providing a porous titanium substrate;

[0013] constructing a TiO2 nanotube array structure on the surface of the porous titanium substrate;

[0014] depositing upconversion nanoparticles;

[0015] growing g-C3N4 nanoparticles in situ by a hydrothermal method;

[0016] filling a hydrogel loaded with carbon nanotubes and a pharmaceutically active ingredient, and immersing in a crosslinking agent solution, to obtain the product.

[0017] Preferably, the TiO2 nanotube array structure is constructed on the surface of the porous titanium substrate by acid etching treatment, anodic oxidation treatment and annealing treatment in sequence; the solvent used in the acid etching treatment is a mixed solution of hydrofluoric acid and nitric acid; the voltage of the anodic oxidation treatment is 20~30 V, and the solvent of the anodic oxidation treatment is an aqueous solution of ammonium fluoride and glycerol; the temperature of the annealing treatment is 400~500℃, and the time is 0.5~2 h.

[0018] Preferably, the step of depositing up-conversion nanoparticles is as follows: dissolving Y salt, Yb salt, Er salt and urea in water to prepare a rare earth solution, then immersing a porous titanium substrate with a surface-constructed TiO2 nanotube array structure in the rare earth solution at 80-95 ℃ for 2-5 h, taking out and immersing in a mixed solution of NaF and HF, and reacting at 95-110 ℃, and then washing and drying.

[0019] Preferably, the step of growing g-C3N4 nanoparticles in situ by a hydrothermal method is as follows: dissolving dicyandiamide and ammonium nitrate in water to prepare a precursor solution, immersing the porous titanium substrate with deposited up-conversion nanoparticles in the precursor solution, and performing a hydrothermal reaction at 160-190 ℃ for 8-15 h, and then washing and drying.

[0020] Preferably, the crosslinking agent solution is a 1-3 wt% calcium salt solution, and the immersion time is 5-15 min; and the preparation method of the hydrogel loaded with carbon nanotubes and a pharmaceutically active ingredient is as follows: dispersing carbon nanotubes and a pharmaceutically active ingredient in water, adding gelatin and sodium alginate thereto, and heating and stirring at 35-50 ℃ to obtain the hydrogel loaded with carbon nanotubes and a pharmaceutically active ingredient.

[0021] In a third aspect, the application provides an application of the above-mentioned light-heat-electricity coupling titanium-based implant or the light-heat-electricity coupling titanium-based implant prepared by the above-mentioned preparation method in the preparation of a bone tissue defect repair material.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] (1) The application realizes the synergistic coupling of light-heat-electricity multi-energy forms by designing a composite structure composed of a porous titanium substrate with a surface having a TiO2 nanotube array, an intermediate functional particle layer containing up-conversion nanoparticles and g-C3N4 nanoparticles, and a hydrogel layer loaded with carbon nanotubes and a pharmaceutically active ingredient, and endows the titanium-based implant with the integrated functions of light response, heat conversion, electric signal conduction and local treatment, thereby providing an integrated solution for bone tissue defect repair with structural support, biological activity regulation and therapeutic effect.

[0024] (2) The TiO2 nanotube array on the surface of the porous titanium substrate not only improves the biocompatibility and surface activity of the substrate, but also acts as a semiconductor light response unit to synergize with Yb 3+ and Er 3+ co-doped NaYF4 up-conversion nanoparticles and g-C3N4 nanoparticles to enhance the light absorption and energy conversion efficiency, and ensure the efficient performance of the light-heat-electricity coupling process.

[0025] (3) The carbon nanotubes loaded in the hydrogel layer can realize effective conduction of thermal-electric signals, and the carbon nanotubes can cooperate with the medicinal active ingredients to realize the dual effects of local physical stimulation and biochemical treatment, further promote the regeneration and repair of bone tissue, and improve the clinical application effect of the implant. BRIEF DESCRIPTION OF DRAWINGS

[0026] The accompanying drawings, which form a part of the present description, are used to provide further understanding of the present application, and the illustrative embodiments thereof, and their descriptions serve to explain the present application, and do not constitute improper limitations on the present application. Obviously, for those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 is a scanning electron microscope picture of Ti-RC-Cur of Example 1 of the present application;

[0028] Figure 2 is an infrared spectrum of Ti-RC-Cur of Example 1 of the present application, Ti of Comparative Example 1 and Ti-RC of Comparative Example 2;

[0029] Figure 3 is a graph of the change of the temperature of the sample surface of the Ti-RC-Cur sample of Example 1 of the present application, the Ti sample of Comparative Example 1 and the Ti-RC sample of Comparative Example 2 with time under the irradiation of near-infrared light (808 nm, 2 W);

[0030] Figure 4 is a graph of the expression of the osteogenesis genes in the rat bone marrow mesenchymal stem cells on the surface of Ti-RC-Cur of Example 1 of the present application and Ti of Comparative Example 1 without near-infrared light treatment (named as Ti-RC-Cur and Ti, respectively) and with near-infrared light treatment (named as Ti-RC-Cur+NIR and Ti+NIR, respectively); wherein, A is a graph of the expression of ALP gene, B is a graph of the expression of COL-1 gene, C is a graph of the expression of OPN gene, and D is a graph of the expression of OCN gene; E is a graph of the expression of Runx2 gene, and F is a graph of the expression of BMP-2 gene; in the graph, the relative expression amount is converted based on the gene expression amount of Comparative Example 1 (set as 1), in the graph, “**” represents P<0.01, “***” represents P<0.001, and “****” represents P<0.0001;

[0031] Figure 5Figure 1 is a graph of surface bacteriostatic rate of Ti-RC-Cur of Example 1 of the present application and Ti of Comparative Example 1 without near-infrared light treatment (named Ti-RC-Cur and Ti, respectively) and with near-infrared light treatment (named Ti-RC-Cur+NIR and Ti+NIR, respectively), wherein A is Escherichia coli and B is Staphylococcus aureus, and in the figure, “****” represents P<0.0001;

[0032] Figure 6 Figure 2 is a graph of cell viability of surface cultured osteosarcoma (HOS) cells after 1d, 2d and 3d of Ti-RC-Cur of Example 1 of the present application and Ti of Comparative Example 1 without near-infrared light irradiation (named Ti-RC-Cur and Ti, respectively) and with near-infrared light irradiation (named Ti-RC-Cur+NIR and Ti+NIR, respectively); in the figure, “ns” represents no significant difference, and “****” represents P<0.0001.

[0033] Figure 7 Figure 3 is a graph of surface current of the light-heat-electricity coupling titanium-based implant of Example 1 of the present application under alternating on and off of near-infrared light with a wavelength of 808nm. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0035] The present application provides a light-heat-electricity coupling titanium-based implant, comprising a porous titanium substrate and a hydrogel layer.

[0036] The surface of the porous titanium substrate is a TiO2 nanotube array structure; upconversion nanoparticles and g-C3N4 nanoparticles are arranged between the porous titanium substrate and the hydrogel layer, the upconversion nanoparticles are Yb 3+ and Er 3+ co-doped NaYF4 upconversion nanoparticles; the hydrogel layer is loaded with carbon nanotubes and a pharmaceutically active ingredient.

[0037] The above-mentioned light-heat-electricity coupling titanium-based implant of the present application realizes efficient coupling and functional integration of light, heat and electricity energy through hierarchical structure design. The TiO2 nanotube array on the surface of the porous titanium substrate serves as a core structural unit, which on the one hand provides stable structural support and biocompatible interface, and on the other hand its semiconductor properties can generate photo-generated charges in response to specific wavelength light. The upconversion nanoparticles (Yb 3+ and Er 3+The co-doped NaYF4) can absorb near-infrared light and convert it into visible light or ultraviolet light through the up-conversion effect, the light response range is expanded, and the short-wave photons generated can be efficiently absorbed by the TiO2 nanotube array and g-C3N4 nanoparticles, solving the problem of insufficient long-wave light absorption of the TiO2 semiconductor material. The g-C3N4 nanoparticles and the TiO2 nanotube array form a heterojunction structure, and the two synergistically promote the separation and transmission of photo-generated charges, reduce charge recombination, and enhance the photoelectric conversion efficiency. At the same time, g-C3N4 and up-conversion nanoparticles further improve the overall light capture capability through light absorption complementation, and together convert part of the light energy into heat energy (photo-thermal effect).

[0038] The carbon nanotubes loaded in the hydrogel layer serve as a key functional medium, which can efficiently conduct the heat energy generated in the middle layer and convert it into an electrical signal through the thermoelectric effect; on the other hand, as an excellent conductive channel, it can quickly transmit the photo-generated current generated by the TiO2 / g-C3N4 heterojunction, realize direct conduction of the photo-electric signal, and promote bone cell differentiation through electrical stimulation; at the same time, the heat conduction property of the carbon nanotubes can uniformly disperse heat, avoiding local overheating. This synergistic mechanism of "up-conversion nanoparticles expanding light absorption → TiO2 / g-C3N4 heterojunction enhancing photo-electric / photo-thermal conversion → carbon nanotube-mediated heat-electric conduction" enables the titanium-based implant to realize multi-path coupling of light, heat and electrical energy. In addition, the medicinal active ingredients in the hydrogel layer can be released controllably under the stimulation of heat / electric signals, and synergistically exert a biological therapeutic effect with the photo-thermal-electric physical stimulation.

[0039] In an optional embodiment of the present application, the material of the hydrogel layer is a gelatin-sodium alginate hydrogel; the gelatin-sodium alginate hydrogel has biocompatibility, degradability and adhesion, and its three-dimensional network structure can stably embed the carbon nanotubes and the medicinal active ingredients.

[0040] In an optional embodiment of the present application, the medicinal active ingredients include curcumin, which has biological activities of anti-inflammatory, antibacterial and promoting osteogenic differentiation, and can be released controllably from the hydrogel network under the stimulation of photo-thermal or electric signals. The medicinal active ingredients can also include other active ingredients such as baicalein. In the hydrogel layer of the present application, the amount ratio of the carbon nanotubes, the medicinal active ingredients, the gelatin and the sodium alginate is (0.05~0.15) μg :(20~50) μg :(0.08~0.12) g :(0.01~0.04) g.

[0041] In an optional embodiment of the present application, in the up-conversion nanoparticles, the doping molar concentration of Yb 3+ is 15~25%, and the doping molar concentration of Er 3+ is 1~5%. Yb 3+ can efficiently absorb near-infrared light and transfer the excitation energy to Er3+ , Er 3+ The near-infrared light with strong tissue penetration is utilized by the visible light or ultraviolet light emitted through the energy level transition, and the problem of low utilization rate of TiO2 on the near-infrared light is solved.

[0042] In an alternative embodiment of the present application, the porous titanium substrate is pure titanium or a titanium alloy, preferably a Ti-6Al-4V alloy, which has both high strength and biocompatibility, while pure titanium is more corrosion-resistant, and both are suitable for different implant environment requirements, and those skilled in the art can select according to actual needs. The porosity of the porous titanium substrate is 50-90%, and the pore size is 500-1000 μm, which is compatible with the pore size of cancellous bone (200-600 μm). The porous titanium substrate is used to simulate the porous microenvironment of natural bone tissue, which on the one hand provides space for bone cell migration, proliferation and vascular ingrowth, and on the other hand provides a rough interface for subsequent surface functionalization and enhances the adhesion of the coating.

[0043] The present application also provides a preparation method of the above-mentioned light-heat-electricity coupling titanium-based implant, comprising the following steps:

[0044] Providing a porous titanium substrate;

[0045] Constructing a TiO2 nanotube array structure on the surface of the porous titanium substrate;

[0046] Depositing up-conversion nanoparticles;

[0047] Growth of g-C3N4 nanoparticles in situ by hydrothermal method;

[0048] Filling the hydrogel loaded with carbon nanotubes and pharmaceutically active ingredients, and soaking in a crosslinking agent solution to obtain.

[0049] In an alternative embodiment of the present application, the TiO2 nanotube array structure is constructed on the surface of the porous titanium substrate by acid etching treatment, anodic oxidation treatment and annealing treatment in sequence; the solvent used in the acid etching treatment is a mixed solution of hydrofluoric acid and nitric acid. The voltage of the anodic oxidation treatment is 20-30 V, and the solvent of the anodic oxidation treatment is an aqueous solution of ammonium fluoride and glycerol. The annealing treatment is carried out at a temperature of 400-500 ℃ for 0.5-2 h; the annealing treatment converts the amorphous TiO2 after anodic oxidation into a crystalline state, which is mainly in the anatase phase, and has high semiconductor activity and chemical stability, ensuring the light response performance of the nanotube.

[0050] In an optional embodiment of the present invention, the steps for depositing upconversion nanoparticles are as follows: A rare earth solution is prepared by dissolving Y salt, Yb salt, Er salt, and urea in water. Then, a porous titanium substrate with a TiO2 nanotube array structure on its surface is immersed in the rare earth solution at 80-95°C for 2-5 hours. After removal, it is immersed in a mixed solution of NaF and HF and reacted at 95-110°C. The substrate is then washed and dried to obtain the final product. In the above preparation method, urea hydrolysis produces OH-. - Make rare earth ions (Y 3+ Yb 3+ Er 3+ A hydroxide precursor is formed on the surface of TiO2 nanotubes; after the addition of NaF and HF, F - Yb is formed by in-situ crystallization with rare earth ions at 95~110℃. 3+ and Er 3+ Co-doped NaYF4 upconversion nanoparticles. During the formation of the rare earth hydroxide precursor, the OH groups on its surface can undergo dehydration condensation with the OH groups on the TiO2 surface, forming Ti-O-RE covalent bonds, thereby anchoring the upconversion nanoparticles to the TiO2 nanotube surface. This invention does not impose special limitations on the Y salt, Yb salt, and Er salt; commonly used Y salts, Yb salts, and Er salts in the preparation of upconversion nanoparticles can be used. Preferably, these are nitrates of Y, Yb, and Er.

[0051] In an optional embodiment of the present invention, the steps of in-situ growth of g-C3N4 nanoparticles via hydrothermal method are as follows: a precursor solution is prepared by dissolving dicyandiamide and ammonium nitrate in water; a porous titanium substrate for depositing upconversion nanoparticles is immersed in the precursor solution; a hydrothermal reaction is carried out at 160-190°C for 8-15 hours; and the substrate is washed and dried to obtain the nanoparticles. Dicyandiamide decomposes into cyanamide under hydrothermal conditions, which polymerizes with ammonium nitrate to generate g-C3N4 nanoparticles. In-situ growth allows g-C3N4 to form a "point-to-point" contact interface with TiO2 and upconversion nanoparticles, shortening the charge transfer path and ensuring that the visible light emitted by upconversion is efficiently absorbed by g-C3N4, followed by rapid transfer of photogenerated charge to TiO2, thus enhancing the charge separation effect of the heterojunction.

[0052] In an optional embodiment of the present invention, the crosslinking agent solution is a 1-3 wt% calcium salt solution, and the soaking time is 5-15 min. The calcium salt solution is further preferably an aqueous solution of calcium chloride, through which calcium ions (Ca...)... 2+) and realize the rapid solidification and structure stability of the hydrogel. The preparation method of the hydrogel loaded with the carbon nanotubes and the pharmaceutically active ingredient is as follows: the carbon nanotubes and the pharmaceutically active ingredient are dispersed in water, the gelatin and the sodium alginate are added, and the hydrogel loaded with the carbon nanotubes and the pharmaceutically active ingredient is obtained by heating and stirring at 35-50 DEG C. The present application does not make special restrictions on the method of filling the hydrogel loaded with the carbon nanotubes and the pharmaceutically active ingredient, and the present application preferably uses a syringe to inject the hydrogel into the porous titanium substrate.

[0053] The present application does not make special restrictions on the preparation method of the porous titanium substrate, and the present application preferably adopts 3D printing. The present application does not make special restrictions on the specific 3D printing method, and the commonly used 3D printing method in the art can be adopted. The present application preferably adopts the selective laser melting (SLM) technology to prepare the porous titanium substrate.

[0054] The present application provides the application of the above-mentioned light-heat-electricity coupling titanium-based implant or the light-heat-electricity coupling titanium-based implant prepared by the above-mentioned preparation method in preparing bone tissue defect repair materials.

[0055] The light-heat-electricity coupling titanium-based implant provided by the present application repairs bone defects through the multi-signal synergistic effect of "light-heat-electricity-biochemistry": the light response component includes upconversion nanoparticles and TiO2 / g-C3N4 heterojunction, which can convert the in-vitro near-infrared light with deep tissue penetration and small damage into an electric signal promoting osteogenesis and a heat signal with antibacterial and metabolic acceleration functions; the carbon nanotubes efficiently conduct the signal to the bone defect area, and the pharmaceutically active ingredient inhibits inflammation and promotes osteogenic differentiation after release; and the porous structure and TiO2 nanotubes synergistically provide structural support and interface conditions for bone integration. The present application breaks through the low repair efficiency limitation of single signals such as only relying on photothermal or only relying on drugs, realizes the integration of "structural support-energy conversion-signal transmission-biological regulation", and is suitable for the repair of complex bone defects such as large segmental bone defects and infected bone defects.

[0056] The technical solutions of the present application will be further described below in combination with specific examples. The present application does not make special restrictions on the sources of reagents used in the following examples, and commercially available goods well known to those skilled in the art can be used.

[0057] In the following examples, the Triply Periodic Minimal Surface (TPMS) is a surface that extends infinitely periodically along three independent directions (such as X, Y, Z axes) in three-dimensional space, and its core characteristic is that the average curvature is zero everywhere. The TPMS structure is highly matched with the zero curvature characteristic of natural bone trabeculae, and the connected channels promote bone cell ingrowth and angiogenesis.

[0058] Example 1

[0059] The present embodiment provides a photo-thermal-electric coupling titanium-based implant and a preparation method thereof.

[0060] (1) Preparation of porous titanium substrate:

[0061] A porous Ti-6Al-4V substrate with a three-periodic minimal surface, a pore size of 700 μm, and a porosity of 70% was prepared using selective laser melting (SLM) technology, and the size was 10 x 10 x 2 mm 3 .

[0062] (2) Construction of TiO2 nanotube array:

[0063] 5 mL of 2 wt% hydrofluoric acid and 31 mL of 20 wt% nitric acid were mixed uniformly to obtain a mixed acid solution, and each surface of the porous Ti-6Al-4V substrate of step (1) was subjected to flow acid etching treatment using the mixed acid solution, and the total treatment time was 90 s. Then, an anodic oxidation treatment was performed in a glycerol containing 0.27 M NH4F at a direct current voltage of 25 V; then, the porous titanium substrate with a TiO2 nanotube array structure on the surface was obtained by annealing at 450°C for 1 h.

[0064] (3) Deposition of upconversion nanoparticles:

[0065] 0.25 g of urea was dissolved in 25 mL of deionized water, and then 250 μL of Y(NO3)3 aqueous solution with a concentration of 1.0 M, 100 μL of Yb(NO3)3 aqueous solution with a concentration of 0.63 M, and 15 μL of Er(NO3)3 aqueous solution with a concentration of 0.4 M were sequentially added. After mixing and dissolving, a rare earth solution was prepared. The porous titanium substrate with a TiO2 nanotube array structure obtained in step (2) was immersed in the rare earth solution at 90°C for 3 h, and then it was immersed in 10 mL of a mixed solution of 0.01 g / mL NaF and 0.03 M HF. After heating at 100°C in a water bath for 30 min, it was taken out, washed with deionized water, and dried to obtain a porous titanium substrate with deposited upconversion nanoparticles.

[0066] (4) In-situ growth of g-C3N4 nanoparticles:

[0067] The porous titanium substrate with deposited upconversion nanoparticles obtained in step (3) was immersed in 10 mL of a precursor aqueous solution containing 0.3 M dicyandiamide and 0.1 M ammonium nitrate, and then hydrothermal reaction was performed at 180°C for 12 h. After washing with deionized water, drying in air was performed to obtain a porous titanium substrate containing g-C3N4 nanoparticles.

[0068] (5) Preparation of hydrogel layer:

[0069] Carbon nanotubes and curcumin were dispersed in water, and the concentration of carbon nanotubes was controlled to be 0.1 μg / mL, and the concentration of curcumin was controlled to be 36.8 μg / mL. Then, gelatin and sodium alginate powders with a mass ratio of 4:1 were added thereto, and the total addition concentration of the two was controlled to be 0.125 g / mL, and then stirring was performed at 45°C for 5 min to obtain a gelatin-sodium alginate hydrogel loaded with carbon nanotubes and curcumin, which was filled into the inside of the porous titanium substrate containing g-C3N4 nanoparticles obtained in step (4) using a syringe, and then was placed in a 2wt% CaCl2 aqueous solution for crosslinking immersion for 10 min, and finally a light-heat-electric coupling titanium-based implant was constructed, which was denoted as Ti-RC-Cur.

[0070] Figure 1 The scanning electron microscope picture of the light-heat-electric coupling titanium-based implant prepared in this example after freeze-drying can be seen that the hydrogel is fully filled in the pores of the porous implant, and the hydrogel after freeze-drying shows a clear porous network structure, forming a porous implant-hydrogel composite.

[0071] Example 2

[0072] The present example provides a light-heat-electric coupling titanium-based implant and a preparation method thereof.

[0073] (1) Preparation of porous titanium substrate:

[0074] A porous Ti-6Al-4V substrate with a three-periodic minimal surface, a pore size of 700 μm, and a porosity of 70% was prepared using selective laser melting (SLM) technology, and the size was 10×10×2 mm 3 .

[0075] (2) Construction of TiO2 nanotube array:

[0076] 5 mL of 2wt% hydrofluoric acid and 31 mL of 20wt% nitric acid were mixed uniformly to obtain a mixed acid solution, and the mixed acid solution was used for flow acid etching treatment of each surface of the porous Ti-6Al-4V substrate of step (1), and the total treatment time was 90 s. Then, anodic oxidation treatment was performed in a propylene glycol containing 0.27 M NH4F at a direct current voltage of 25 V; and then annealing at 450°C for 1 h to obtain a porous titanium substrate with a TiO2 nanotube array structure on the surface.

[0077] (3) Deposition of upconversion nanoparticles:

[0078] A rare earth solution was prepared by dissolving 0.25 g of urea in 25 mL of deionized water, and then sequentially adding 250 μL of a Y(NO3)3 aqueous solution with a concentration of 1.0 M, 100 μL of a Yb(NO3)3 aqueous solution with a concentration of 0.63 M, and 15 μL of an Er(NO3)3 aqueous solution with a concentration of 0.4 M. After mixing and dissolving, the solution was obtained. The porous titanium substrate with the surface-constructed TiO2 nanotube array structure obtained in step (2) was immersed in the rare earth solution at 90°C for 3 h. After being taken out, the substrate was immersed in 10 mL of a mixed solution of 0.01 g / mL NaF and 0.03 M HF, and was heated in a 100°C water bath for 30 min. After being taken out, the substrate was washed with deionized water and dried, to obtain a porous titanium substrate with upconversion nanoparticles deposited thereon.

[0079] (4) In-situ growth of g-C3N4 nanoparticles:

[0080] The porous titanium substrate with upconversion nanoparticles deposited thereon obtained in step (3) was immersed in 10 mL of a precursor aqueous solution containing 0.45 M dicyandiamide and 0.15 M ammonium nitrate, and then was hydrothermally reacted at 180°C for 12 h. After being washed with deionized water, the substrate was dried in air, to obtain a porous titanium substrate containing g-C3N4 nanoparticles.

[0081] (5) Preparation of a hydrogel layer:

[0082] The carbon nanotubes and curcumin were dispersed in water, and the concentration of the carbon nanotubes was controlled to be 0.1 μg / mL, and the concentration of the curcumin was controlled to be 36.8 μg / mL. Then, gelatin and sodium alginate powders with a mass ratio of 4:1 were added thereto, and the total addition concentration of the two was controlled to be 0.125 g / mL. Then, the mixture was stirred at 45°C for 5 min, to obtain a gelatin-sodium alginate hydrogel loaded with carbon nanotubes and curcumin. The hydrogel was injected into the inside of the porous titanium substrate containing g-C3N4 nanoparticles obtained in step (4) by using a syringe, and then was immersed in a 2 wt% CaCl2 aqueous solution for crosslinking for 10 min, to finally construct a light-heat-electricity coupling titanium implant.

[0083] Comparative Example 1

[0084] This comparative example is different from Example 1 in that the anodization treatment of step (2) and steps (3) to (5) are not performed. The finally obtained porous titanium substrate is denoted as Ti.

[0085] Comparative Example 2

[0086] This comparative example is different from Example 1 in that step (5) is not performed. The finally obtained porous titanium substrate with upconversion nanoparticles and g-C3N4 nanoparticles deposited thereon is denoted as Ti-RC.

[0087] Comparative Example 3

[0088] Compared with Example 1, the difference in this comparative example is that step (4) was not performed. In this comparative example, the gelatin-sodium alginate hydrogel loaded with carbon nanotubes and curcumin was directly injected into the porous titanium substrate with deposited upconversion nanoparticles obtained in step (3), and then placed in a 2wt% CaCl2 aqueous solution for crosslinking for 10 min. The titanium-based implant obtained was denoted as Ti-Re.

[0089] Test case

[0090] 1. Infrared measurement

[0091] Infrared spectra of Ti-RC-Cur from Example 1, Ti from Comparative Example 1, and Ti-RC from Comparative Example 2 were measured. Figure 2 As shown, it can be seen that on the Ti group surface that has only undergone acid etching treatment, only obvious Ti-O bonds (961.98 cm⁻¹) can be observed. -1 This indicates that the surface was oxidized in air. In the Ti-RC group, however, significant out-of-plane bending vibrations of the triazine ring units (860.25 cm⁻¹) were observed. -1 This proves that g-C3N4 was successfully added to the surface. In the Ti-RC-Cur group, due to the addition of the hydrogel and the curcumin encapsulated within it, a significant presence of -OH groups was detected (3279.34 cm⁻¹). -1 Meanwhile, the NH in the hydrogel (1538.39cm) -1 ) and -COO (1412.72cm -1 It can be clearly detected. The results of infrared spectroscopy indicate that curcumin was successfully introduced into the titanium-based implant of Example 1.

[0092] 2. Photothermal conversion performance measurement:

[0093] The porous titanium implants of Examples 1-2 and Comparative Examples 1-3 were irradiated with near-infrared light (NIR) at a wavelength of 808 nm and a power of 2 W for 5 min, and the surface temperature of the samples was measured. The results are summarized in Table 1. The surface temperature rise of the Ti-RC-Cur sample of Example 1, the Ti sample of Comparative Example 1, and the Ti-RC sample of Comparative Example 2 are as follows: Figure 3 As shown.

[0094] Table 1. Surface temperature of porous titanium implants in Examples 1-2 and Comparative Examples 1-3 after 5 min of near-infrared light irradiation.

[0095]

[0096] As can be seen from Table 1, Comparative Example 1 showed almost no obvious photothermal properties, and the temperature was close to room temperature. For Examples 1, 2, and 3, since g-C3N4 has no obvious photothermal properties, both groups showed similar temperature change characteristics, remaining at around 54.2℃. However, for Comparative Example 2, due to the lack of photothermal properties from carbon nanotubes, the temperature decreased slightly to 52.6℃.

[0097] 3. Expression of osteogenic-related genes in rat bone marrow mesenchymal stem cells:

[0098] The expression of osteogenic genes in rat bone marrow mesenchymal stem cells was detected using RT-qPCR (reverse transcription real-time quantitative polymerase chain reaction). Rat bone marrow mesenchymal stem cells were processed at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / mL onto the surface of Ti-RC-Cur samples from Example 1 and Ti samples from Comparative Example 1. The experimental groups of Example 1 and Comparative Example 1, namely the Ti-RC-Cur+NIR group and the Ti+NIR group, were treated with near-infrared light (NIR), and were irradiated with 808 nm wavelength and 2 W power for 10 min daily during culture. After 7 days of culture, cells were washed with PBS buffer, and 600 μL of Trizol reagent was added to each well. Subsequently, RNA was reverse transcribed into cDNA using a reverse transcription kit. The expression of six target genes (ALP, OCN, COL-I, BMP-2, OPN, and Runx2) and the internal control gene GAPDH was tested. Gene expression was normalized according to GAPDH expression, and the test results are shown below. Figure 4 As shown.

[0099] from Figure 4 As shown in Figures A through F, after near-infrared light irradiation, the expression level of the OCN gene in the Ti-RC-Cur+NIR group was 1.86 times that of the Ti group. The expression levels of ALP, OPN, Runx2, BMP-2, and COL-I all reached more than twice that of the Ti group, indicating that the expression of osteogenic-related genes significantly increased under surface modification and near-infrared light irradiation, and the upregulation of early osteogenic differentiation genes such as ALP was more pronounced.

[0100] 4. Antibacterial performance test:

[0101] The antibacterial rate of the sample surface was quantitatively determined by measuring the bacterial absorbance values ​​on the surface of the porous titanium implants of Examples 1 and Comparative Examples 1-3.

[0102] The test method is as follows: the bacteria are inoculated on the surface of the sample, and after incubation at 37°C for 24 h, they are transferred to a new well without bacteria, and the surface is rinsed with sterile culture medium and the original solution is diluted 10 times. After that, the diluted solution is incubated at 37°C for 24 hours, and the absorbance of each well is detected at 600 nm using an enzyme marker. For the near-infrared light irradiation group, near-infrared light with a wavelength of 808 nm and a power of 2 W is used for irradiation for 10 min per day. The bacteriostatic rate of the surface of each group of samples is calculated according to the following formula:

[0103]

[0104] The test results are summarized in Table 2.

[0105] Table 2 Bacteriostatic rate of porous titanium implant of Example 1 and Comparative Examples 1-3

[0106]

[0107] Figure 5 The surface bacteriostatic rate results of Ti-RC-Cur of Example 1 and Ti of Comparative Example 1 are shown in the figure, where A is Escherichia coli and B is Staphylococcus aureus. From Table 2 and Figure 5 It can be seen that in Example 1, the bacteriostatic rate for Escherichia coli and Staphylococcus aureus is 54.55% and 52.71% without near-infrared light irradiation, and the surface bacteriostatic rate reaches about 89% with near-infrared light irradiation, significantly inhibiting bacterial growth. For Comparative Example 1, the surface shows almost no significant bacteriostatic rate. For Comparative Example 2, the surface shows poor bacteriostatic rate without near-infrared light irradiation, only about 13%, which shows that the introduction of hydrogel containing carbon nanotubes and curcumin can further improve the antibacterial effect; after the addition of near-infrared light irradiation, the bacteriostatic rate increases to about 49%. In Comparative Example 3, the bacteriostatic rate is only about 40% without the addition of near-infrared light irradiation, and after the addition of near-infrared light irradiation, the electric signal is significantly weakened due to the non-addition of g-C3N4, and the bacteriostatic rate is only about 66%.

[0108] 5. Osteosarcoma cell viability assay:

[0109] Osteosarcoma (HOS) cells were seeded at 5 x 10 4The density of 1*104 / mL was inoculated on the surface of the Ti-RC-Cur of Example 1 and the Ti sample of Comparative Example 1. The culture groups with light-heat (Ti+NIR and Ti-RC-Cur+NIR) were irradiated with near-infrared light with a wavelength of 808 nm and a power of 2 W for 10 min every day during the culture process. After 1, 2 and 3 days of culture, the samples were transferred to new sample wells, and 1 mL of fresh culture medium and 100 μL of CCK-8 solution were added to the sample wells. After incubation at 37 °C for 1 h, 200 μL of the solution was transferred to a 96-well plate, and the absorbance of the sample was detected using an enzyme-labeled instrument, and then the cell viability was calculated. The formula for calculating the cell viability is as follows:

[0110]

[0111] The experimental group refers to the culture group containing the porous titanium implant sample, and the blank culture medium refers to the culture medium without the porous titanium implant sample.

[0112] The results are shown in Table 1. Figure 6 As can be seen, there is no obvious difference between the groups at 1 day of culture. However, at the second day, the cell viability changes obviously. The cell viability of the Ti-RC-Cur group decreases to 76.3%, and after the addition of light-heat treatment, the cell viability of the Ti-RC-Cur+NIR group decreases to 50%. At the third day, this trend is more obvious. The cell viability of the Ti-RC-Cur group decreases to 54%, and the cell viability of the Ti-RC-Cur+NIR group decreases to 22%.

[0113] 6. Surface photoelectric performance test

[0114] The surface of the light-heat-electricity coupling titanium-based implant of Example 1 was irradiated with near-infrared light with a wavelength of 808 nm, and the current of the surface was detected using an electrochemical workstation. The results are shown in Table 2. Figure 7 During the experiment, the light was turned on and off alternately for 20 s, and it can be observed that when the light is turned on, there is obvious photoelectric current on the surface, and when the light is turned off, the photoelectric current on the surface decreases obviously, indicating that the surface of the light-heat-electricity coupling titanium-based implant of Example 1 has obvious photoelectric response characteristics, which is conducive to stimulating the osteogenic differentiation of mesenchymal stem cells and the death of bacteria through the current effect, and the photoelectric current will produce reactive oxygen, causing oxidative stress in the cells, causing damage to the lipids, proteins and DNA in the cells, thereby promoting cell apoptosis.

[0115] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A photo-thermal-electric coupled titanium-based implant, characterized in that, Includes a porous titanium substrate and a hydrogel layer; The porous titanium substrate has a TiO2 nanotube array structure on its surface; the hydrogel layer is made of gelatin-sodium alginate hydrogel; upconversion nanoparticles and g-C3N4 nanoparticles are disposed between the porous titanium substrate and the hydrogel layer, wherein the upconversion nanoparticles are Yb 3+ and Er 3+ Co-doped NaYF4 upconversion nanoparticles; the hydrogel layer is loaded with carbon nanotubes and pharmaceutically active ingredients; A TiO2 nanotube array structure was constructed on the surface of a porous titanium substrate by sequentially performing acid etching, anodizing, and annealing treatments; the annealing treatment was carried out at a temperature of 400~500℃ for a time of 0.5~2h. The preparation method of the g-C3N4 nanoparticles is as follows: dicyandiamide and ammonium nitrate are dissolved in water to prepare a precursor solution, a porous titanium substrate with deposited upconversion nanoparticles is immersed in the precursor solution, and a hydrothermal reaction is carried out at 160~190℃ for 8~15h. After washing and drying, g-C3N4 nanoparticles grown in situ by hydrothermal method are obtained. In the hydrogel layer, the ratio of carbon nanotubes, pharmaceutical active ingredients, gelatin, and sodium alginate is (0.05~0.15)μg : (20~50)μg : (0.08~0.12)g : (0.01~0.04)g; The photo-thermal-electric coupled titanium-based implant repairs bone defects through the synergistic effect of multiple signals including photo-thermal-electrical-biochemical processes. The method for preparing the photo-thermal-electric coupled titanium-based implant includes the following steps: Provide porous titanium substrates; Constructing a TiO2 nanotube array structure on a porous titanium substrate; Deposition of upconversion nanoparticles; g-C3N4 nanoparticles were grown in situ using a hydrothermal method. The hydrogel filled with carbon nanotubes and pharmaceutically active ingredients is obtained by soaking it in a crosslinking agent solution.

2. The photo-thermal-electric coupled titanium-based implant as described in claim 1, characterized in that, The medicinal active ingredient includes curcumin.

3. The photo-thermal-electric coupled titanium-based implant as described in claim 1, characterized in that, In the upconversion nanoparticles, Yb 3+ The doping molar concentration is 15~25%, Er 3+ The doping molar concentration is 1~5%.

4. The photo-thermal-electric coupled titanium-based implant as described in claim 1, characterized in that, In the method for preparing the photo-thermal-electric coupled titanium-based implant, a TiO2 nanotube array structure is constructed on the surface of a porous titanium substrate by sequentially performing acid etching, anodizing, and annealing treatments; the solvent used for the acid etching treatment is a mixed solution of hydrofluoric acid and nitric acid; the voltage for the anodizing treatment is 20~30V, and the solvent for the anodizing treatment is an aqueous solution of ammonium fluoride and glycerol.

5. The photo-thermal-electric coupled titanium-based implant as described in claim 1, characterized in that, In the preparation method of the photo-thermal-electric coupled titanium-based implant, the step of depositing upconversion nanoparticles is as follows: a rare earth solution is prepared by dissolving Y salt, Yb salt, Er salt and urea in water, and then a porous titanium substrate with a TiO2 nanotube array structure on the surface is immersed in the rare earth solution at 80~95℃ for 2~5h. After being taken out, it is immersed in a mixed solution of NaF and HF and reacted at 95~110℃. After washing and drying, the implant is obtained.

6. The photo-thermal-electric coupled titanium-based implant as described in claim 1, characterized in that, In the preparation method of the photo-thermal-electric coupled titanium-based implant, the crosslinking agent solution is a 1-3 wt% calcium salt solution, and the soaking time is 5-15 min; the preparation method of the hydrogel loaded with carbon nanotubes and pharmaceutical active ingredients is as follows: the preparation method of the hydrogel loaded with carbon nanotubes and pharmaceutical active ingredients is as follows: carbon nanotubes and pharmaceutical active ingredients are dispersed in water, gelatin and sodium alginate are added to it, and the mixture is heated and stirred at 35-50°C to obtain the hydrogel loaded with carbon nanotubes and pharmaceutical active ingredients.

7. The application of the photo-thermal-electric coupled titanium-based implant as described in any one of claims 1 to 6 in the preparation of bone tissue defect repair materials.

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