CN113403619A

CN113403619A - near-infrared/pH dual-response iodine-loaded titanium alloy implant and preparation method thereof

Info

Publication number: CN113403619A
Application number: CN202110623028.0A
Authority: CN
Inventors: 叶招明; 滕王锶源; 俞小华; 刘安; 章增杰; 王翊凯; 汪方乾
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-09-17

Abstract

The invention discloses a near-infrared/pH dual-response iodine-carrying titanium alloy implant system with antibacterial and osteogenic differentiation promoting functions and a preparation method thereof. The surface coating titanium sheet prepared by the invention can show good antibacterial performance under the excitation of weak acidity and near infrared light, and can promote osteogenic differentiation of bone marrow mesenchymal stem cells under a normal physiological state. The iodine coating prepared by the invention has the advantages of good stability and controllable release, and effectively solves the problem of insufficient stability of the prior metal surface iodine modification.

Description

near-infrared/pH dual-response iodine-loaded titanium alloy implant and preparation method thereof

Technical Field

The invention relates to the technical field of orthopedics, oral and maxillofacial surgery and neurosurgery implants, in particular to a near-infrared/pH dual-response iodine-carrying titanium alloy implant with antibacterial and osteogenesis promoting functions and a preparation method thereof.

Background

With the increased use of orthopedic endoprosthesis instruments in the last decade, endoprosthesis infection has become an increasingly serious clinical problem. Once infection occurs, it often means at least a treatment period of months and a very large second revision surgery, which will cause great pain and burden to the patient himself and the family. Debridement and antibiotic therapy (DAIR) with preservation of joint prostheses is considered the first choice for treatment of prosthetic infections. However, the complex debridement environment, the generation of drug-resistant bacteria, and the remaining bacterial residues on the surface of the prosthesis pose a potential for early infection treatment failure. Thus, imparting antimicrobial properties to the endoprosthesis itself is a non-antibiotic solution against prosthesis infection.

Currently, researchers have developed a range of antibacterial materials that impart antibiotic-independent antibacterial properties to medical implants. The formulation of these materials typically involves the incorporation of metallic elements, peptides or polymers, etc. By utilizing the characteristics of the materials, the physical damage of cells can be directly caused or the physiological processes of bacteria, such as DNA replication, cell membrane permeability and the like, can be specifically interrupted. For example, the material is modified by utilizing the property that silver ions can destroy the cell membrane of bacteria and eliminate the contact with the bacteria, so that the medical implant material with the antibacterial property is constructed. Although these strategies have shown encouraging efficacy against bacterial infections and largely avoided the abuse of antibiotics, on the other hand, the main drawback is that the introduction of such metallic element-modified endoprostheses is a relatively new field of exploration, which implies difficulties in terms of certification and supervision as well as in finding the optimal therapeutic window, and the difficulty of combining effective antibacterial properties without cytotoxic effects, which largely limits their clinical application. Therefore, it is necessary to find more powerful and relatively safe coating modifications.

Povidone iodine is the most widely used disinfectant in clinical practice. Iodine is a micronutrient associated with energy metabolism and neurodevelopment and plays a key role in the use of povidone-iodine to combat pathogenic bacterial invasion. Iodine/iodine compounds can effectively kill bacteria and eradicate established biofilms by disrupting cellular substructures and interfering with expression of genetic material. To date, no clinical cases of iodine-induced drug-resistant bacteria have been reported, making iodine an excellent candidate for replacing antibiotics. Although iodine has been widely used as a disinfectant for hundreds of years, few studies have reported the use of iodine to combat infection of orthopedic implants in the body. There have been studies showing that the use of iodine based disinfectants internally significantly enhances the antimicrobial properties of these internal implant systems. However, due to the insoluble and unstable nature of iodine in solution, iodine release cannot be controlled because of the limited iodine loading caused by the simple physical adsorption of iodine solution on the titanium surface. And since iodine tends to sublimate into iodine vapor, this makes the method vulnerable to damage to surrounding tissue under physiological conditions. Therefore, new ideas and methods are needed to achieve successful modification of iodine in inner plant materials.

In recent years, Metal Organic Frameworks (MOFs) have attracted considerable attention by researchers because ordered metal nanostructures provide ultra-large surface areas, high loading capacities, well-defined porous structures, and controllable degradation behavior. By selecting proper organic ligand and reaction conditions, the MOF structure formed on the surface of the material can be used as a drug-carrying and controlled-release carrier to promote various medical applications. In particular, MOFs have great potential to combat bacterial infections because of their composition/structure-related characteristics, including responsive degradation with bacterial activity, generation of reactive oxygen species, and ability to carry other antibacterial agents. In addition to these advantages, ZIF-8 has particular advantages in the antibacterial field due to its high photocatalytic efficiency, responsiveness to acidic pH values involved in bacterial infections, low cytotoxicity, etc. It is envisioned that ZIF-8 may be an excellent candidate for MOF-based antimicrobial surface modification in the context of PJI. In addition, the ZIF-8 material has extremely strong adsorption performance to iodine elementary substance, which also enables the material to be used as a surface modification material for carrying iodine.

In the invention, a composite coating with near infrared/pH dual-response explosive iodine release and ZIF-8 photodynamic effect is constructed. The dual-response control system is constructed by fixing ZIF-8 on the surface of micro-arc titanium oxide through in-situ hydrothermal treatment, and then loading iodine on the ZIF-8 by utilizing the high absorption capacity of a ZIF-8 coating and adopting a vapor deposition process. After the system is stimulated by acidic or near-infrared irradiation for a certain time, the iodine loaded on the ZIF-8 can be rapidly released in a triggering burst mode due to the degradation of the ZIF-8 carrier, and then the strong antibacterial effect of the iodine is exerted. We demonstrate that the modified materials exhibit significantly improved antimicrobial performance both in vitro and in vivo when the material system is exposed to an infectious environment and to NIR excitation. Furthermore, triggered release of iodine was successfully achieved by using NIR laser as a "switch" for iodine delivery, which substantially solved the weaknesses of previous methods of iodine fixation. In addition, the system can also induce osteogenic differentiation of human mesenchymal stem cells, and endows the material with the characteristics of resisting bacteria and promoting osteogenic differentiation.

Disclosure of Invention

In order to solve the problem that the prosthesis is easy to be infected by bacteria, the invention provides a near infrared/pH dual-response iodine-carrying titanium alloy implant system with antibacterial and bone differentiation promoting functions and a preparation method thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a near-infrared/pH dual-response iodine-carrying titanium alloy implant, which takes a titanium alloy base material with a porous structure formed by surface micro-arc oxidation as a matrix, introduces a zeolite imidazole ester framework structure (ZIF-8) nanoparticle coating on the surface of the matrix and inside a pore channel through hydrothermal reaction, adopts a vapor deposition method to adsorb iodine vapor on the coating, and finally removes impurities through high-temperature high-pressure steam treatment to finally obtain a stable iodine-carrying coating system.

Specifically, the near-infrared/pH dual-response iodine-loaded titanium alloy implant is prepared by the following method:

(a) after pretreatment, immersing the titanium alloy base material into an electrolytic cell for electrochemical treatment for 3-30min (preferably 5min) to obtain the titanium alloy base material (porous film structure) containing the coating; the electrochemical treatment is anodic oxidation or micro-arc oxidation, the voltage is maintained at 500V (preferably 150V) of 100-;

the aperture of the matrix film obtained by the electrochemical means of the micro-arc oxidation is 2-10 mu m, and the density is 10000 holes/mm²And a base coating layer having a thickness in the range of 50-80 μm.

(b) Immersing the titanium alloy substrate containing the coating obtained in the step (a) in a reaction solution, placing the titanium alloy substrate in a reaction kettle with a polytetrafluoroethylene inner container, treating the titanium alloy substrate in the reaction kettle with a high-temperature reaction furnace at the temperature of 120-150 ℃ for 6-10 hours (preferably treating the titanium alloy substrate at the temperature of 120 ℃ for 6 hours), washing the titanium alloy substrate with pure water for three times, placing the titanium alloy substrate in an oven at the temperature of 60-150 ℃ for drying (removing residual liquid on the surface of the material, preferably drying the titanium alloy substrate at the temperature of 60 ℃) to obtain the titanium alloy substrate with the ZIF-8 nano particle coating; the reaction liquid is prepared from a zinc nitrate aqueous solution and a 2-methylimidazole aqueous solution, and the mass ratio of zinc nitrate contained in the zinc nitrate aqueous solution to 2-methylimidazole contained in the 2-methylimidazole aqueous solution is 1:1-80 (preferably 1: 80); the mass of zinc nitrate contained in the zinc nitrate aqueous solution is 0.5-2.0 wt% (preferably 0.67%) of the reaction solution;

(c) putting the titanium alloy substrate of the ZIF-8 nano-particle coating obtained in the step (b) and elemental iodine into a closed reaction container in a non-contact manner (performing iodine vapor deposition treatment); heating at 40-70 deg.C for 1-6 hr (preferably 60 deg.C for 3 hr); taking out and washing for 3 times by using pure water (removing unadsorbed iodine simple substance on the surface); carrying out sterilization treatment to obtain the near-infrared/pH dual-response iodine-loaded titanium alloy implant; the mass of the elementary iodine is not less than 6mg/mL of the volume of the reaction container, but not more than the mass of the elementary iodine is in contact with the titanium alloy substrate of the ZIF-8 nano-particle coating.

Further, the pretreatment in the step (a) is mainly an impurity removal step, and the operation flow is as follows: and washing the titanium alloy base material with acetone, ethylene glycol and ultrapure water in sequence to remove impurities, then carrying out acid washing to remove impurities, and finally carrying out ultrasonic cleaning and then taking out.

In the pretreatment process in the step (a), the mass concentration of acetone is more than or equal to 99.5%, and the mass concentration of ethylene glycol is more than or equal to 99.5%.

The solution used in the pickling process in step (a) consists of the following components in mass concentration: 0.8 to 2% of HF, 5.2 to 5.44% of HNO₃，1.275％H₃PO₄The solvent is water, and the acid washing time is 5-25min (preferably 15 min);

in the step (a), the titanium alloy base material is fixed on the anode side (fixed by a titanium alloy hook support or a mesh basket), and the cathode is a pure titanium plate or a lead plate.

And (b) in the step (a), the ultrasonic cleaning is to place the titanium alloy base material after acid cleaning in ultrapure water for 5 min/time, and the circulation is carried out for 3 times.

Preferably, the acid electrolytic cell in the step (a) uses sulfuric acid (with a final concentration of 35g/L) -phosphoric acid (with a final concentration of 25g/L) -hydrogen peroxide (with a final concentration of 10g/L) -water system as an electrolyte, and the alkaline electrolytic cell uses potassium hydroxide (with a final concentration of 165g/L) -potassium fluoride (with a final concentration of 35g/L) -sodium phosphate (with a final concentration of 35g/L) -aluminum hydroxide (with a final concentration of 35g/L) -water system as an electrolyte.

More preferably, in the step (b), the volume ratio of the zinc nitrate aqueous solution to the 2-methylimidazole aqueous solution is 1:10, the zinc nitrate aqueous solution is prepared from zinc nitrate hexahydrate and pure water in a mass ratio of 169:1155, and the 2-methylimidazole aqueous solution is prepared from 2-methylimidazole and pure water in a mass ratio of 1093: 385.

Further, the sterilization treatment in step (c) is: and (3) placing the titanium alloy base material subjected to the iodine vapor deposition treatment into an autoclave, and treating for 20-30 minutes (preferably 20 minutes) at the temperature of 121 ℃ and under the condition of 103.4 kPa.

The iodine-carrying titanium alloy coating system can be used for preparing orthopedic, oral and maxillofacial surgery and neurosurgery implants, and specifically comprises the following components: the titanium alloy external fixation frame bone nail, the artificial joint prosthesis, the tumor type prosthesis, the personalized additive manufacturing (3D printing) titanium alloy series joint prosthesis and the like.

The iodine-loaded titanium alloy coating has the characteristics of anti-infection property and osteogenic differentiation promotion under the double regulation of near infrared/pH, and the release amount of iodine in the prepared iodine-loaded system coating can be obviously increased under the condition of weak acidity/acidity (pH is 6.0 and below) or long-time excitation of near infrared light (time is 10 minutes or more), so that the material antibacterial property is presented, and the effect of controllable antibacterial performance of the material is achieved. The anti-infection is mainly aimed at common bacteria of orthopedic infection, including gram-positive bacteria and gram-negative bacteria, wherein the gram-positive bacteria include staphylococcus aureus, drug-resistant staphylococcus aureus, staphylococcus epidermidis, drug-resistant staphylococcus epidermidis and the like.

Compared with the prior art, the invention has the beneficial effects that:

1. the iodine-loaded titanium alloy implant obtained by modifying the titanium alloy implant by using the iodine coating not only has the function of resisting infection, but also has the function of promoting osteogenic differentiation of mesenchymal stem cells. Wherein the bacteriostatic property of the coating is dual regulated and controlled by near infrared/pH. The invention can effectively overcome the clinical defects of the prior titanium alloy implant applied to orthopedics, oral and maxillofacial surgery and neurosurgery, reduce the incidence rate of postoperative early infection after the titanium alloy implant is implanted into a body and cause the related complications such as implant loosening and the like.

2. The invention utilizes the super-strong adsorption performance of materials such as ZIF-8 on iodine and simultaneously utilizes the principle of vapor deposition to stably load iodine simple substances on a ZIF-8 coating. The iodine elementary substance loaded and combined by the ZIF-8 has the advantages of stable property, high-temperature treatment resistance and adjustable release rate, and the obtained iodine-loaded titanium alloy implant has very obvious effects on the aspects of antibiosis and bone differentiation promotion.

Drawings

FIG. 1 Electron microscope observation of the material of example 1 shows the uniform porous structure of the surface (MAO) after micro-arc oxidation; ZIF-8 coating (MAO + Z) and iodine-loaded ZIF-8 coating (MAO + ZI).

Figure 2X-ray diffraction (XRD) results for the material of example 1.

FIG. 3 Fourier transform Infrared absorption Spectroscopy (FTIR) results for the materials of example 1.

Fig. 4 material cross-section spectroscopy analysis (EDXS) in example 1 suggested that iodine was bound to the ZIF-8 coating (zinc coating) scale bar at 100 μm.

FIG. 5 iodine loading of the material after autoclaving and degradation and release rates of ZIF-8 coating and iodine in different pH environments for example 1. (A) Carrying iodine on two substrate materials, and carrying iodine amount after removing impurities at high temperature; (B) release amount of iodine element; (C) release amount of zinc element

FIG. 6 the change in iodine release rate of the material coating in example 1 with and without near infrared stimulation.

FIG. 7 is a test of the bacteriostatic properties of the material coating in vitro in example 1.

FIG. 8 in vitro cytotoxicity assay of material layers in example 1.

FIG. 9 in vitro osteoinductive differentiation experiment of material coating in example 1, hBMSC cell osteogenesis related gene expression.

FIG. 10 results of Micro-CT experiments on in-vivo animal osteogenic repair of materials in example 2.

FIG. 11 shows the electron microscope observation results of the coating before and after loading iodine in different processes in the comparative example.

Detailed description of the preferred embodiments

The invention will be further described by means of specific embodiments with reference to the attached drawings, but the scope of protection of the invention is not limited thereto.

EXAMPLE 1 preparation of iodine-carrying titanium alloy flakes

1. Construction of porous matrix film on surface of titanium sheet

A porous titanium alloy substrate with the roughness Ra of 0-0.4 mu m and the size of 8 multiplied by 2mm (provided by Zhejiang medical instruments Co., Ltd., the porous titanium alloy substrate is convenient for supporting a bracket and the front and back surfaces of a metal sheet are fully contacted with reaction liquid), and the porous titanium alloy substrate is pretreated by acetone (more than or equal to 99.5 percent), glycol (more than or equal to 99.5 percent), ultrapure water and the like in sequence, and then the material is placed into a reaction kettle containing HF (more than or equal to 40 percent and 20ml/L) and HNO (HNO)₃(65.0～68.0％，80ml/L)、H₃PO₄Acid washing in solution system (85%, 15ml/L) for 15min, finally ultrasonic cleaning for 3 times, and blow drying.

The treated titanium alloy material is subjected to micro-arc oxidation operation, and the method comprises the following specific steps: dissolving 140mL of 95.0-98.0 wt% sulfuric acid (CAS #7664-93-9, national drug group chemical agents Co., Ltd.), 100mL of 85 wt% phosphoric acid (Aladdin, CAS #7664-38-2) and 40mL of 30-31% hydrogen peroxide (Aladdin, CAS #7722-84-1) in water to prepare 4L of electrolyte, fixing the material on an anode side titanium alloy hook, wherein a cathode is a pure titanium plate with the thickness of 3mm, the distance between the anode side and the cathode side is 10-20cm, and the reaction conditions are as follows: the voltage is 150V, the frequency is 1000HZ, the water temperature is controlled at 30-50 ℃, and the reaction time is 5 min; and finally, placing the sample subjected to the oxidation operation in an aqueous solution for ultrasonic immersion cleaning for 3min, and repeating the cleaning operation for 3 times.

2. Introduction of ZIF-8 coatings on substrate films

And preparing a reaction solution. First, 2.3mL of reaction solution A in which 0.338g of zinc nitrate hexahydrate (1.1mmol) was dissolved in water and 23.1mL of reaction solution B in which 6.558g of 2-methylimidazole (80mmol) was dissolved in water were prepared. After the A, B reaction solution is completely dissolved, slowly dripping the solution A into the solution B in a magnetic stirring state, and fully stirring for 10min until the mixed solution is milky white. The mixture was transferred to a 50mL autoclave with a Teflon liner.

And (3) selecting a polytetrafluoroethylene support frame with a proper size, uniformly placing 9 substrates treated in the step (1) on the polytetrafluoroethylene support frame, and immersing the substrates in the mixed liquid of the reaction kettle.

The reaction kettle is placed in an oven at 120 ℃ for 6 hours and taken out. The base material is washed for 3 times by pure water and is dried in a drying oven at 60 ℃.

3. Iodine vapor deposition and high temperature impurity removal

Putting each piece of material processed in the step 2 and about 15mg of elementary iodine particles into a closed reaction container with the capacity of 2mL, and keeping a non-contact state; the sealed container was placed in a 60 ℃ oven and heated for 3 hours. Subsequently, the material was taken out and washed 3 times with pure water. And finally, placing the material subjected to the iodine vapor deposition treatment into a high-pressure steam sterilization pot, and treating for 20 minutes at the temperature of 121 ℃ and under the condition of 103.4kPa to remove unadsorbed iodine simple substances on the surface and finish the sterilization process.

4. Surface appearance and structure of iodine-carrying titanium alloy coating system

Fig. 1 shows the material observed by electron microscopy, showing: a porous structure with uniform surface (MAO) after micro-arc oxidation; modifying the surface uniform particle crystal by a ZIF-8 coating (MAO + Z); the surface crystal of the iodine-loaded ZIF-8 coating (MAO + ZI) is changed from a long-short ordered structure into a short-sequence structure after adsorbing iodine. FIG. 2 shows the formation of ZIF-8 structure on MAO + Z surface by submitting the material to XRD; after the MAO + ZI carries iodine on the surface, the crystal deforms, and the characteristic peak disappears. FIG. 3FTIR suggests that ZIF-8 coatings of materials (MAO + Z and MAO + ZI) are stable before and after loading with iodine. FIG. 4EDXS suggests that iodine is bound to the ZIF-8 coating (zinc containing coating). The thickness of the micro-arc oxidation matrix film is about 80 mu m.

5. The high temperature resistance of the iodine-carrying titanium alloy coating system.

FIG. 5.A shows that MAO + ZI can still stably bind part of the iodine after autoclaving compared to a coating with a simple porous structure. The operation steps are as follows: and (3) treating the material subjected to the simple micro-arc oxidation and the material introduced with the ZIF-8 coating in the step (3), then soaking and washing the material in an ultrasonic cleaning machine by using 30 wt% of nitric acid, and detecting the concentration of iodine element in the soaking and washing solution through an inductively coupled plasma mass spectrometer (ICP-MS).

6. The pH value of the iodine-carrying titanium alloy coating system regulates and controls the iodine release characteristic.

FIGS. 5, B and C show the release of iodine and zinc from MAO + ZI coating materials in acidic and neutral environments. The operation steps are as follows: the MAO + ZI coated piece was completely immersed in 2mL SBF solution at pH 6.0 and pH 7.4, respectively, and the immersion solution was completely changed at specific time points (3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 6 days, 9 days). And detecting the concentrations of the zinc element and the iodine element of the collected leachate by ICP-MS.

7. The near infrared of the iodine-carrying titanium alloy coating system regulates and controls the iodine release characteristic.

First, the MAO + ZI material piece was immersed in 2ml SBF for 1 hour to ensure that the ions started to release stably. Then, 0/10min near infrared irradiation and 30min standing treatment were performed in this order. During which the solution was replaced thoroughly every 20 minutes. The above 40 minute period served as one cycle. The release rate of iodine is calculated by the following formula:

iodine release rate-iodine concentration (μ g/mL)/sampling frequency time (20 min) × 100%

8. In-vitro antibacterial test of iodine-carrying titanium alloy coating

Bacteriostatic experiments of the iodine-carrying titanium alloy implant: the antibacterial detection of the invention adopts staphylococcus aureus (ATCC 25923) as experimental bacteria, two groups of titanium sheets, namely MAO titanium sheet and MAO + ZI titanium sheet in the operation are disinfected and then placed in a 24-hole plate, and each group is provided with 3 multiple holes. Staphylococcus aureus (ATCC 25923) bacterial liquid (about 2-5X 106CFU/ml) was inoculated to each of the above titanium sheets in an amount of 100. mu.L per well, and the resultant was subjected to static culture at 37 ℃ for 18 hours in a constant humidity environment. Bacteria adhered to the surface of the titanium sheet are eluted by sterile PBS, the eluent is diluted to a proper concentration in an equal proportion, and then an agarose gel culture dish is used for coating, and after the agarose gel culture dish is cultured for 24 hours in an incubator at 37 ℃, colonies are counted and photographed.

FIG. 7 is the in vitro antibacterial performance test of different material coatings.

9. In vitro cytotoxicity test of iodine-carrying titanium alloy coating

In the research, human-derived mesenchymal stem cells (hBMSCs) are used as experimental cells, but the research is not limited to hBMSCs, and various cells of other species are all suitable for the research.

Cytotoxicity was measured using CCK-8 detection kit (Dojindo, Japan). 500 μ L of hBMSCs at 5X 10⁴Cells/well were plated onto 48-well plates and incubated for 3 and 7 days. At day 3 and day 7 time points, media was discarded and was gently rinsed 3 times with PBS. To each well was added 220. mu.L of a mixed solution of the culture solution and CCK-8 kit (v/v 10: 1). Finally, after incubation at 37 ℃ for 2h, the absorbance (OD) was measured at a wavelength of 450 nm.

FIG. 8 is an in vitro cytotoxicity assay with different material layers.

10. In vitro bone induction differentiation experiment of iodine-carrying titanium alloy coating

Selecting each group of titanium sheets, wherein the diameter is 20mm, and the height is 2 mm. Placing in 6-hole plate after sterilization by wet heat method, each group is provided with 3 multiple holes. Suspension of hBMSCs cells (1.0X 10)⁵Per well) was seeded on the surface of the material in an amount of 5mL per well. The medium used in this step was osteogenic differentiation induction medium (containing 10% fetal bovine serum, 1% glutamine and 1% sodium beta-glycerophosphate, 400. mu.L ascorbic acid and 20. mu.L dexamethasone, Seikagaku (Guangzhou) Bio Inc.). The culture medium was changed every two days. Total RNA from hBMSCs attached to the surface of different samples was extracted by RNAioso plus (Takara Co.) and reverse transcribed by PrimeScript TM RT kit (EZBioscience Co.) at 7 and 14 days. And (3) performing cyclic amplification by adopting a two-step method. The parameters were that the reverse transcription samples were kept at 95 ℃ for 30s, and then 42 cycles of keeping at 95 ℃ for 5s and 60 ℃ for 30s were started. The primer sequences for the different genes are shown in Table 1. GADPH was used as the internal reference frame.

TABLE 1

FIG. 9 shows the change of expression level of osteogenesis related genes after culturing two groups of titanium plate surface cells. Research can obtain that the iodine-carrying coating system can better induce osteogenic differentiation of hBMSCs compared with a simple micro-arc oxidation surface.

EXAMPLE 2 preparation of titanium-iodine-carrying alloy rod

1. Construction of porous matrix film on surface of titanium sheet

A titanium alloy rod base material (provided by Zhejiang medical instrument Co., Ltd.) with the diameter of 1.2mm and the length of 10mm is adopted to be sequentially pretreated by acetone (not less than 99.5%), ethylene glycol (not less than 99.5%), ultrapure water and the like, then the material is put into a solution system containing HF (not less than 40%, 20ml/L), HNO3 (65.0-68.0%, 80ml/L) and H3PO4 (85%, 15ml/L) for acid cleaning for 15min, and finally ultrasonic cleaning is carried out for 3 times and blow drying is carried out.

The treated titanium alloy material is subjected to micro-arc oxidation operation, and the method comprises the following specific steps: dissolving 140mL of 95.0-98.0 wt% sulfuric acid (CAS #7664-93-9, national medicine group chemical reagents Co., Ltd.), 100mL of 85 wt% phosphoric acid (Aladdin, CAS #7664-38-2) and 40mL of 30-31% hydrogen peroxide (Aladdin, CAS #7722-84-1) in water to prepare 4L of electrolyte, placing the materials in a titanium alloy mesh basket connected with an anode, wherein the cathode is a pure titanium plate with the thickness of 3mm, the distance between the anode side and the cathode side is 10-20cm, and the reaction conditions are as follows: the voltage is 150V, the frequency is 1000HZ, the water temperature is controlled at 30-50 ℃, and the reaction time is 5 min; and finally, placing the sample subjected to the oxidation operation in an aqueous solution for ultrasonic immersion cleaning for 3min, and repeating the cleaning operation for 3 times.

2. Introduction of ZIF-8 coatings on substrate films

And preparing a reaction solution. First, 2.3mL of reaction solution A in which 0.338g of zinc nitrate hexahydrate was dissolved in water and 23.1mL of reaction solution B in which 6.558g of water was dissolved were prepared. After the A, B reaction solution is completely dissolved, slowly dripping the solution A into the solution B in a magnetic stirring state, and fully stirring for 10min until the mixed solution is milky white. The mixture was transferred to a 50mL autoclave with a Teflon liner.

And (3) selecting and putting 18 titanium rods treated in the step (1) into the mixed solution of the reaction kettle.

3. Iodine vapor deposition and high temperature impurity removal

Placing the material treated in the step (2) and excessive elemental iodine particles (the concentration is more than or equal to 6mg/mL) into a closed reaction container, and keeping a non-contact state; the sealed container was placed in a 60 ℃ oven and heated for 3 hours. Subsequently, the material was taken out and washed 3 times with pure water. And finally, placing the material subjected to the iodine vapor deposition treatment into a high-pressure steam sterilization pot, and treating for 20 minutes at the temperature of 121 ℃ and under the condition of 103.4kPa to remove unadsorbed iodine simple substances on the surface and finish the sterilization process.

4. In vivo osseointegration effect of iodine-carrying coating material

8-week-old male SD rats are selected to be injected with 1.5 wt% of pentobarbital sodium whole anesthesia and local lidocaine hydrochloride for anesthesia. These rats were randomly divided into four groups. The tibia was prepared, the tibia was exposed, and each set of titanium rods was placed at the proximal end of the tibia. After surgery 28, 56d, 3 rats per group were sacrificed by injection of excess chloral hydrate. The tibia was fixed with 4% paraformaldehyde. And (5) conveying the sample to Micro-CT for detection.

Comparative example

The ZIF-8 coating on the surface of the titanium alloy material is mostly carried out by adopting a hydrothermal treatment method on the surface subjected to alkali heat treatment. In order to obtain the titanium metal surface subjected to alkali heat treatment, the method comprises the following steps: the TC4 alloy was ultrasonically cleaned 3 times with acetone and deionized water, respectively, to prepare Alkaline Heating Titanium (AHT). Then, etching was performed with 4 wt% hydrofluoric acid for 2 minutes, and then washing was performed with deionized water to completely remove impurities. After cleaning, the material was soaked in a Teflon-lined autoclave containing sodium hydroxide solution at a concentration of 5mol/L and heated to 60 ℃ for 24 h. After cooling, the titanium plate was rinsed three times with deionized water at room temperature and reacted at 37 ℃ for 24 hours. Finally, the prepared flat plate is heated in a high temperature furnace at the heating rate of 5 ℃/min and is kept at 600 ℃ for 1 hour.

However, we found that the coating bonding strength was poor on the surface after the alkali heat treatment, and electron microscopy after iodine loading and impurity removal treatment suggested that most of the surface ZIF-8 nanoparticles had disappeared (FIG. 11). According to the invention, a porous structure formed by micro-arc oxidation of the surface of a titanium alloy is used as a matrix, a zeolite imidazole ester framework (ZIF-8) nanoparticle coating is introduced on the surface of the matrix and inside a pore channel through hydrothermal reaction, iodine vapor is adsorbed on the coating by adopting a vapor deposition method, and finally, impurities are removed through high-temperature high-pressure steam treatment, so that a stable iodine-loaded coating system is finally obtained. The stability of iodine loaded on the surface is guaranteed, the controllable release of the loading components is designed, the experimental verification of clinical conversion of medical instruments is facilitated, and a bridge which is guided from a laboratory to clinic is built.

Claims

1. A near-infrared/pH dual-response iodine-carrying titanium alloy implant is characterized in that the near-infrared/pH dual-response iodine-carrying titanium alloy implant is prepared by the following method:

(a) after pretreatment, immersing the titanium alloy base material into an electrolytic cell for electrochemical treatment for 3-30min to obtain the titanium alloy base material containing the coating; the electrochemical treatment is anodic oxidation or micro-arc oxidation, the anode is the titanium alloy base material, the voltage is maintained at 500V for 100-;

(b) immersing the titanium alloy substrate containing the coating obtained in the step (a) in a reaction solution, treating the titanium alloy substrate in a reaction kettle with a polytetrafluoroethylene inner container at the temperature of 120-150 ℃ for 6-10 hours by a high-temperature reaction furnace, washing the titanium alloy substrate by pure water, and drying the titanium alloy substrate in a drying oven at the temperature of 60-150 ℃ to obtain the titanium alloy substrate with the ZIF-8 nano-particle coating; the reaction solution is prepared from a zinc nitrate aqueous solution and a 2-methylimidazole aqueous solution, wherein the mass ratio of zinc nitrate contained in the zinc nitrate aqueous solution to 2-methylimidazole contained in the 2-methylimidazole aqueous solution is 1: 1-80; the mass of zinc nitrate contained in the zinc nitrate aqueous solution is 0.5-2.0 wt% of the reaction solution;

(c) placing the titanium alloy substrate of the ZIF-8 nano-particle coating obtained in the step (b) and elemental iodine into a closed reaction container in a mutual non-contact manner; heating at 40-70 deg.C for 1-6 hr; taking out and washing with pure water; carrying out sterilization treatment to obtain the near-infrared/pH dual-response iodine-loaded titanium alloy implant; the mass of the elementary iodine is not less than 6mg/mL of the volume of the reaction container, but not more than the mass of the elementary iodine is in contact with the titanium alloy substrate of the ZIF-8 nano-particle coating.

2. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: the pretreatment in the step (a) is as follows: and washing the titanium alloy base material with acetone, ethylene glycol and ultrapure water in sequence to remove impurities, then carrying out acid washing to remove impurities, and finally carrying out ultrasonic cleaning and then taking out.

3. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 2, wherein: the solution used for pickling consists of the following components in mass concentration: 0.8-2% of HF, 5.2-5.44% of HNO₃And 1.275% H₃PO₄The solvent is water.

4. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: in the step (a), the acidic electrolytic cell uses sulfuric acid-phosphoric acid-hydrogen peroxide-water system as electrolyte, and the alkaline electrolytic cell uses potassium hydroxide-potassium fluoride-sodium phosphate-aluminum hydroxide-water system as electrolyte.

5. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 4, wherein: the electrolyte in the acid electrolytic cell consists of the following components in final concentration: 35g/L sulfuric acid, 25g/L phosphoric acid and 10g/L hydrogen peroxide; the electrolyte of the alkaline electrolytic cell consists of the following components in final concentration: 165g/L potassium hydroxide, 35g/L potassium fluoride, 35g/L sodium phosphate and 35g/L aluminum hydroxide.

6. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: the conditions of the electrochemical treatment in the step (a) are that the voltage is 150V, the current frequency is 1000Hz, and the temperature of the electrolyte in the electrolytic cell is 30-50 ℃.

7. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: the cathode of the electrochemical treatment is a pure titanium plate or a lead plate.

8. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: the parameters of the high-temperature reaction furnace in the step (b) are 120 ℃ for 6 hours.

9. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: in step (c), the reaction vessel was heated at 60 ℃ for 3 hours.

10. The near-infrared/pH dual-responsive iodine-loaded titanium alloy implant of claim 1, wherein: the sterilization treatment in the step (c) is as follows: and (3) placing the titanium alloy base material subjected to the iodine vapor deposition treatment into a high-pressure steam sterilization pot, and treating for 20-30 minutes at the temperature of 121 ℃ under the condition of 103.4 kPa.