Selenium-containing high molecular compound modified zinc alloy material and preparation method and use thereof

Abstract

The application belongs to the technical field of biomedical engineering, and relates to a selenium-containing high polymer compound modified zinc alloy material and a preparation method and application thereof. The selenium-containing high polymer compound modified zinc alloy material is obtained by coordination and compounding of a selenium-containing high polymer compound and a zinc alloy, and the selenium-containing high polymer compound is prepared from raw materials including H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate and polyethylene glycol monomethyl ether through a reaction. The selenium-containing high polymer compound modified zinc alloy material prepared by the preparation method can be used as an oral implant or an orthopedic implant, and can reduce inflammation caused by implantation after being implanted into a body, and realize the synergistic effect of anti-inflammation and bone formation promotion.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical engineering, and relates to a zinc alloy material modified by a selenium-containing high molecular compound and a preparation method and application thereof. BACKGROUND

[0002] Oral and maxillofacial large-area bone defect has various causes, a high incidence, a complex shape, and a great difficulty in bone regeneration, which brings great challenges to clinical treatment. The current gold standard for treatment is bone transplantation or biologically inert metal bone tissue engineering scaffolds. Commonly used titanium alloy scaffolds have high strength, excellent corrosion resistance, and mature processing technology, but cannot be degraded, and in many cases need to be removed by secondary surgery, which has the risk of reinfection and increases the time and economic burden of doctors and patients. Therefore, it is necessary to explore a degradable metal that can provide mechanical support in the early stage of implantation and can be gradually degraded and absorbed in the body as the bone regeneration process progresses. The human body contains various trace elements, and the current research on biodegradable alloys mainly focuses on magnesium (Mg), iron (Fe), and zinc (Zn). Among them, magnesium alloy generates hydrogen during the degradation process, and the degradation rate is too fast, resulting in the loss of strength in the body too early; iron alloy has a slow corrosion rate and needs several years to completely degrade.

[0003] Zinc, as an essential trace element in the human body, has advantages such as high initial mechanical strength, appropriate degradation rate, and promotion of osteogenesis. Its standard electrode potential (-0.763 V) is between that of Mg (-2.372 V) and Fe (-0.447 V), which has good application prospects. However, the burst release of zinc in the early stage of degradation can cause cytotoxicity in vitro and delay bone integration in vivo, affecting its biocompatibility and potential applications.

[0004] To improve this problem, the main methods currently used are alloying and surface modification. Alloying can control the release speed of ions while improving the mechanical properties of the alloy. Compared with alloying, surface modification only changes the surface properties of the metal, maintaining the original properties of the bulk material, and forming a functional temporary surface that can resist bacteria and promote bone formation.

[0005] There are related patents that focus on using alloying and surface modification of zinc to improve its corrosion resistance, such as the patent application "Zinc-based medical material containing metal / zinc phosphate composite coating and its preparation method and application" (Application No. CN202310427212.7), which uses an ion deposition method to prepare a layer of iron metal on the surface of a zinc substrate, and then uses a chemical method to prepare a composite coating of zinc phosphate to achieve uniform corrosion and controllable corrosion rate. However, the biological characterization of the medical material obtained by this method is not perfect, and only the degradation efficiency has been preliminarily verified.

[0006] Selenium, an essential trace element for the human body, participates in functions such as anti-inflammation, anti-oxidation, antibacterial activity, and accelerating heavy metal detoxification. Low concentrations of selenium act as antioxidants by selenoproteins scavenging reactive oxygen species (ROS), while excessive selenium generates oxygen free radicals, inducing apoptosis. These beneficial properties make selenium-containing compounds applicable in the treatment of tumors, diabetes, and cardiovascular diseases. Current research focuses on using selenium-containing compounds as release carriers on the surfaces of non-degradable metals such as titanium. For example, the patent application "Bioactive Artificial Joint Capable of Slowly Releasing Trace Element Selenium" (application number: CN201210015430.1) modifies the titanium surface with a surface layer loaded with trace amounts of sodium selenite and bone morphogenetic proteins. However, further research has not yet explored the effects of such modified layers on inhibiting ion burst release in degradable zinc alloys or on promoting osteogenic processes. Summary of the Invention

[0007] The primary objective of this invention is to provide a zinc alloy material modified with selenium-containing polymer compounds, which can reduce implant-induced inflammation after implantation and achieve a synergistic effect of anti-inflammatory and bone-promoting properties when used as an oral implant or orthopedic implant.

[0008] To achieve this objective, in a basic implementation, the present invention provides a zinc alloy material modified with a selenium-containing polymer compound. The zinc alloy material modified with the selenium-containing polymer compound is obtained by coordination compounding of a selenium-containing polymer compound with a zinc alloy. The selenium-containing polymer compound is prepared by reaction from raw materials containing H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate, and polyethylene glycol monomethyl ether.

[0009] In a preferred embodiment, the present invention provides a zinc alloy material modified with a selenium-containing polymer compound, wherein the zinc alloy is selected from zinc-lithium alloys and / or zinc-lithium-magnesium alloys, preferably Zn0.8Li and / or Zn0.8Li0.4Mg.

[0010] The second objective of this invention is to provide a method for preparing zinc alloy materials modified with selenium-containing polymers as described above, so as to better prepare zinc alloy materials modified with selenium-containing polymers as described above. The resulting zinc alloy materials modified with selenium-containing polymers can be used as dental implants or orthopedic implants, reducing implant-induced inflammation after implantation and achieving a synergistic effect of anti-inflammatory and bone-promoting action.

[0011] To achieve this objective, in a basic embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, the preparation method comprising the following steps:

[0012] (1) Preparation of selenium-containing polymers: H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate, polyethylene glycol monomethyl ether and catalyst are mixed in an organic solvent and reacted. The product obtained from the reaction is recrystallized and washed.

[0013] (2) Dissolve the selenium-containing polymer compound obtained in step (1) in an organic solvent, and then soak it in a zinc alloy for modification reaction.

[0014] In a preferred embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, wherein in step (1),

[0015] The organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, and n-butyl acetate;

[0016] The diisocyanate is selected from one or more of toluene 2,4-diisocyanate, hexamethylene diisocyanate, diphenylmethane diisocyanate, and isophorone diisocyanate;

[0017] The molar ratio of H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate, and polyethylene glycol monomethyl ether is x:1:(1.1x+1.1):(0.2x+0.2), where x is 0.1 to 10;

[0018] The catalyst is dibutyltin dilaurate.

[0019] In a preferred embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, wherein in step (1), the reaction temperature is 30-100℃ and the reaction time is 1-48 hours.

[0020] In a preferred embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, wherein in step (1), the recrystallization is carried out by recrystallization with diethyl ether, and the washing is carried out by washing with ethanol at a volume percentage concentration of 5-50%.

[0021] In a preferred embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, wherein in step (2),

[0022] The organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide;

[0023] The concentration of selenium-containing polymers dissolved in organic solvents is 1-10 mg / mL.

[0024] In a preferred embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, wherein in step (2),

[0025] The zinc alloy is a zinc alloy sheet or a zinc alloy rod;

[0026] The zinc alloy is polished and ultrasonically cleaned before use.

[0027] In a preferred embodiment, the present invention provides a method for preparing a zinc alloy material modified with a selenium-containing polymer compound as described above, wherein in step (2), the modification reaction takes 1-3 hours.

[0028] A third objective of this invention is to provide the use of the zinc alloy material modified with selenium-containing polymer compounds as described above for the preparation of dental implants or orthopedic implants, so as to reduce implant-induced inflammation after implantation and achieve a synergistic effect of anti-inflammatory and bone-promoting action.

[0029] To achieve this objective, in a basic embodiment, the present invention provides the use of the zinc alloy material modified with selenium-containing polymer compounds as described above for the preparation of dental implants or orthopedic implants.

[0030] The beneficial effect of the present invention is that the zinc alloy material modified with selenium-containing polymer compounds prepared by the preparation method of the present invention can be used as a dental implant or orthopedic implant, and can reduce implant-induced inflammation after implantation, thereby achieving a synergistic effect of anti-inflammatory and bone-promoting.

[0031] This invention modifies the surface of biodegradable zinc alloys with selenium-containing polymer compounds through chemical bonding. Utilizing the coordination between selenium and zinc and the wear-resistant properties of polyurea compounds, it achieves efficient and reliable modification of biodegradable zinc alloys. This modification can alleviate the wear of Zn... 2+ This invention addresses the biotoxicity issues of existing zinc alloys both in vivo and in vitro, expanding their clinical applications. When the selenium-modified zinc alloy material of this invention is implanted into the body, the effects of selenium on surrounding tissues can reduce implantation-induced inflammation and effectively promote osteogenic formation of mesenchymal stem cells (hBMMSCs). It also induces THP-1-derived macrophages to polarize towards the anti-inflammatory M2 type and inhibits their polarization towards the pro-inflammatory M1 type. This enables the multifunctionality of the implant material, achieving a synergistic effect of anti-inflammatory and osteogenic properties, and its applicability in disease treatment, while promoting bone regeneration around the implant. Attached Figure Description

[0032] Figure 1The image shows the characterization results of the selenium-containing small molecule obtained in Example 1, where a is the structural formula of the selenium-containing small molecule; b is the molecular weight result of the selenium-containing small molecule determined by electrospray mass spectrometry; and c is the nuclear magnetic resonance hydrogen spectrum, selenium spectrum, and carbon spectrum of the selenium-containing small molecule.

[0033] Figure 2 The figures show the structural formulas of the selenium-containing polymers prepared in Example 1 and Comparative Example 1, and the characterization results of their successful modification on biodegradable zinc alloys. Specifically, a) shows the structural formulas of the selenium-containing polyurea polymer SePUA prepared in Example 1 and the selenium-containing polyurethane polymer SePU prepared in Comparative Example 1; b) shows the 1H NMR spectrum of the selenium-containing polyurea polymer prepared in Example 1; c) shows the gel permeation chromatography characterization results of the selenium-containing polyurea polymer prepared in Example 1; d) shows the contact angle diagram of the biodegradable zinc alloy surface modified with the selenium-containing polymer prepared in Example 1; and e) shows the selenium ion surface scan of the zinc alloy surface obtained by second-time-of-flight ion mass spectrometry.

[0034] Figure 3 The following images illustrate the effect of the selenium-containing polymer compound modified with the biodegradable zinc alloy prepared in Example 1 on delaying the degradation of the zinc alloy: a) shows the weight loss of the alloy during the degradation process; b) shows the pH change of the degradation solution; and c) shows the surface microstructure of the biodegradable zinc alloy after degradation under an electron microscope before and after modification with the selenium-containing polymer compound.

[0035] Figure 4 The results of biocompatibility testing for the selenium-containing polymer-modified biodegradable zinc alloys prepared in Example 1 and Comparative Example 1 are shown, where a represents the Zn content in the 24-hour leaching solution of the selenium-containing polymer-modified zinc alloys and the unmodified zinc alloys prepared in Example 1 and Comparative Example 1. 2+ Concentration; b is the CCK-8 result of hBMMSCs cultured with different concentrations of degradable zinc alloy extract; c is the cell uptake result of selenium in human bone marrow mesenchymal stem cells (hBMMSCs) cultured in vitro with the selenium-containing polymer-modified zinc alloy prepared in Example 1; d is the live / dead cell staining result of the degradable zinc alloy extract prepared in Example 1 before and after modification with different proportions of hydrophilic groups and different concentrations of selenium-containing polymer compounds.

[0036] Figure 5The images show the effect of selenium-containing polymers modified with biodegradable zinc alloys prepared in Example 1 and Comparative Example 1 on promoting osteogenic activity of human bone marrow mesenchymal stem cells (hBMMSCs) in vitro. A and B represent the alkaline phosphatase (ALP) staining and quantification results of hBMMSCs in unmodified zinc alloys and zinc alloys modified with different concentrations of selenium-containing polymers from the examples and comparative examples. C shows the expression detection results of osteogenic-related genes OSX and OCN by qPCR. D shows the expression detection results of osteogenic-related protein RUNX2 by Western blotting. E shows the immunofluorescence images of hBMMSCs cultured in extracts of biodegradable zinc alloys before and after modification with selenium-containing polymers.

[0037] Figure 6 The images show the polarization of THP-1-derived macrophages cultured in vitro using selenium-containing polymers modified with biodegradable zinc alloys prepared in Example 1 and Comparative Example 1. Figure a shows the expression results of related genes iNOS and ARG-1 detected by qPCR; figure b shows the expression results of inflammation-related proteins iNOS and ARG-1 detected by Western Blot; figure c shows the intracellular reactive oxygen species (ROS) level detection results; and figure d shows the expression results of polarization-related proteins iNOS and ARG-1 detected by immunofluorescence.

[0038] Figure 7 The in vivo osteogenic effect of the selenium-containing polymer modified biodegradable zinc alloy prepared in Example 1 and Comparative Example 1 on femoral defects in SD rats after implantation is shown in Figure a, where a is the result of Micro-CT scan and b is the result of hard tissue grinding and staining. Detailed Implementation

[0039] The following examples further illustrate specific embodiments of the present invention.

[0040] Example 1: Preparation and Characterization of Selenium-Containing Polymers (Part 1)

[0041] 1.0 g of selenium powder (0.0127 mol) and 2.0 g of sodium borohydride (0.0529 mol) were added to a 250 mL round-bottom flask. 30 mL of water was slowly added dropwise under ice-water bath conditions. After the reaction gradually slowed and gas production ceased, the flask was sealed with a rubber stopper containing a balloon, yielding a colorless and transparent solution. The solution was then allowed to return to room temperature for later use. 5.6 g of 3-bromo-1-propylamine hydrobromide (0.0254 mol) was dissolved in 10 mL of water. Under stirring, the prepared 3-bromo-1-propylamine hydrobromide solution was added to the colorless and transparent solution using a syringe and needle. The reaction was allowed to proceed for 24 h at room temperature. After the reaction was complete, dichloromethane and deionized water were added to the remaining mixture. The liquid-liquid phase was separated and the organic phase was collected. The aqueous phase was washed with dichloromethane until colorless, and the organic phase solutions were combined. Anhydrous sodium sulfate was added and dried. The anhydrous sodium sulfate was removed by filtration. Most of the dichloromethane was removed by rotary evaporation to obtain a concentrated solution. The solution was then dried under vacuum to obtain a pale yellow-white liquid. The resulting selenium-containing small molecule H2N-(CH2)3-Se-(CH2)3-NH2 has the following molecular structure: Figure 1 As shown in Figure a, the results of electrospray mass spectrometry determination of the molecular weight of selenium-containing small molecules are as follows: Figure 1 As shown in b, the 1H NMR, selenium, and carbon NMR spectra of selenium-containing small molecules are as follows: Figure 1 As shown in c.

[0042] 1,4-Di(2-hydroxyethyl)piperazine and selenium-containing small molecules H2N-(CH2)3-Se-(CH2)3-NH2, along with 1.3 mmol of 2,4-diisocyanate (2,4-TDI), were added to 8 mL of tetrahydrofuran at different molar ratios as shown in Table 1. 20 μL of dibutyltin dilaurate was then added. The mixture was sealed with a rubber stopper, and nitrogen gas was purged into the system for 5 min to remove air from the reaction apparatus. The reaction was then heated to 90 °C for 5 h. After the reaction was complete, the resulting product was precipitated dropwise in vigorously stirred ice-cold ether. The precipitate was collected by filtration and washed five times with a 10% (v / v) ethanol aqueous solution, and then lyophilized to remove water. The purified polymer was analyzed by 1H NMR spectroscopy (results are shown in [Table 1]). Figure 2 b) and gel permeation chromatography (results are shown in...) Figure 2 c) Characterization was performed, and the results showed that the desired selenium-containing polymer compound (structure shown in [see structure]) was successfully synthesized. Figure 2 a).

[0043] Table 1

[0044]

[0045] Comparative Example 1: Preparation and Characterization of Selenium-Containing Polymers (Part 2)

[0046] Selenium-containing polymeric compounds were prepared and characterized according to Examples 1-2 of Chinese Patent Application No. 202410220052.3.

[0047] Example 2: Preparation and characterization of zinc alloy materials modified with selenium-containing polymer compounds

[0048] (1) Preparation of zinc alloy materials modified with selenium-containing polymer compounds

[0049] Weigh 50 mg of the selenium-containing polymer compound prepared in Example 1 and dissolve it in 5 mL of dimethyl sulfoxide (DMSO) to obtain a clear and transparent solution. Prepare a 10 mg / mL selenium-containing polymer solution. Based on the ratio of hydrophilic groups to selenium groups in the structure of the selenium-containing polymer compound, prepare three selenium-containing polymer solutions: 75% Se (hydrophilic group: selenium group = 1:3), 50% Se (hydrophilic group: selenium group = 1:1), and 25% Se (hydrophilic group: selenium group = 3:1). According to experimental requirements, further dilute the selenium-containing polymer solutions with different selenium group ratios with DMSO to three concentrations of 1 mg / mL, 3 mg / mL, and 5 mg / mL for subsequent experiments.

[0050] A Zn0.8Li alloy (containing 0.8 wt% Li and 99.2 wt% Zn) was prepared. 99.99% pure metallic Zn and 99.9% pure metallic Li were first melted at 520℃, then mixed and cast into a cylindrical shape. The zinc alloy was cut into Φ20mm×0.5mm and Φ7mm×0.5mm sheets and Φ1.5mm×5mm rods using molybdenum wire cutting. Before use, all zinc alloys were progressively polished with silicon carbide sandpaper to 10000 grit to obtain a smooth, oxide-free surface. They were then ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and deionized water for 10 minutes each to remove surface residues. After removal, the material was patted dry and then immersed for 2 hours in selenium-containing polymer solutions of different concentrations and selenium group ratios for surface modification. After removal, the material was rinsed with anhydrous ethanol, air-dried, and placed in appropriately sized perforated plates for later use. For ease of description, the zinc alloy modified with selenium-containing polyurea polymer of Example 1 prepared according to this method will be abbreviated as SePUA-Zn0.8Li in the following text, and the proportion of selenium groups in the selenium-containing polymer used for modification and the concentration of the solution will be indicated. Unless otherwise specified, it is modified with 25% Se 3mg / mL selenium-containing polymer.

[0051] Similarly, the zinc alloy material modified with selenium-containing polyurethane polymer prepared in Comparative Example 1 was prepared. Hereinafter, the zinc alloy modified with selenium-containing polyurethane will be referred to as SePU-Zn0.8Li, and the proportion of selenium groups in the selenium-containing polymer and the concentration of the solution will be indicated. Unless otherwise specified, the modification was performed using 25% Se 3 mg / ml selenium-containing polymer.

[0052] (2) Detection of the surface modification effect of zinc alloy materials modified with selenium-containing polymer compounds

[0053] The static contact angles of biodegradable zinc alloy surfaces before modification and after modification with different concentrations of selenium-containing polymers were measured using a static contact angle meter (Dataphysics, Germany) to compare their hydrophilicity. Each group of pre-prepared biodegradable zinc alloy sheets was placed horizontally on the sample stage and adjusted to a suitable position. A deionized water droplet was slowly released from the needle, ensuring the droplet formed a spherical shape upon contact with the sample surface. The camera parameters were adjusted, and the meter's imaging system captured images of the contact angle between the droplet and the solid surface. The volume and velocity of the added droplet were kept constant each time, and the droplet morphology and contact angle were measured. To ensure the reliability of the results, five test points were randomly selected for each group, and the average contact angle was calculated as the final result. Results are as follows: Figure 2 As shown in d, it can be seen that the selenium-containing polyurea of ​​Example 1 has better hydrophilicity after modification on the zinc alloy surface than the selenium-containing polyurethane of Comparative Example 1 after modification on the zinc alloy surface, and the hydrophilicity decreases with the increase of the proportion of selenium groups in the selenium-containing polymer.

[0054] The surfaces of biodegradable zinc alloys (represented as Zn-0.8Li, SePUA-Zn0.8Li, and SePU-Zn0.8Li, respectively) before and after modification with selenium-containing polyurea and selenium-containing polyurethane were analyzed using time-of-flight secondary ion mass spectrometry (ION-TOF GmbH, Germany). The results are shown in the figure. Figure 2 As shown in e, it can be seen that the selenium-containing polymers prepared in Example 1 and Comparative Example 1 can be uniformly modified on the metal surface, and the concentration of Se on the metal surface after modification with selenium-containing polyurea in Example 1 is significantly higher.

[0055] Example 3: Degradability Test of Zinc Alloy Materials Modified with Selenium-Containing Polymer Compounds

[0056] Zinc alloy sheets with a diameter of 7 mm and a thickness of 0.5 mm were polished stepwise with 500-10000 grit sandpaper. After polishing, the experimental group's zinc alloy was placed in a 25% Se 3 mg / mL selenium-containing polymer solution (Example 1) for 2 hours for modification. The control group consisted of unmodified zinc alloy sheets. Three samples were collected from each group at each time point. The samples from both the experimental and control groups were immersed in simulated body fluid (SBF, purchased from Beijing Coollab Technology Co., Ltd., catalog number SL6710), with a solution volume to zinc alloy surface area ratio of 20 mL / cm². 2 The system was placed in a shaker at 37°C. Zinc alloy samples were removed at 1, 3, 5, 7, 14, 21, 28, 42, and 56 days, and the simulated body fluid was collected and replaced. The zinc alloy sheets were then rinsed sequentially with 200 g / L CrO3, deionized water, and anhydrous ethanol to remove surface degradation products. After drying in an oven at room temperature for 2 hours, the samples were weighed using an electronic balance. The weight changes of the samples are shown below.Figure 3 As shown in Figure a, during the first few days of immersion, the zinc alloy modified with selenium-containing polymers in Example 1 showed a significantly slower corrosion process compared to the unmodified group. Throughout the degradation process, the weight loss of the modified group was less than that of the corresponding unmodified group at all time points, indicating that the modification with selenium-containing polymers can slow down the degradation rate of the zinc alloy, especially slowing down the early burst release of zinc alloy degradation. The pH values ​​of the solutions collected at the above time points were measured, and the results are as follows. Figure 3 As shown in b, the extraction solution becomes more alkaline after modification with selenium-containing polymers, which is more conducive to osteogenic differentiation of hBMMSCs.

[0057] After collecting samples at day 56 of degradation, the microstructure and corrosion morphology of the selenium-modified and unmodified polymer surfaces were observed using a scanning electron microscope (JSM-7900F, Japan). The results are as follows: Figure 3 As shown in c, the particulate degradation products are more uniformly distributed on the zinc alloy surface after modification with selenium-containing polymers compared to the unmodified zinc alloy surface.

[0058] Example 4: In vitro biocompatibility testing of zinc alloy materials modified with selenium-containing polymer compounds

[0059] The biodegradable zinc alloy leachate was prepared according to ISO 10993 standard. The specific method is as follows: Φ20mm × 0.5mm zinc alloy metal discs (before and after modification with selenium-containing polymers, as described in Example 1) were immersed in an aqueous solution with a specific surface area of ​​1.25 mL / cm². 2 The cell proliferation medium (PM: α-MEM + 10% FBS + 1% penicillin-streptomycin) was used for extraction on a shaker at 37°C for 24 hours, followed by filtration using a sterile filter in a laminar flow hood. The extract was mixed with PM at different volume ratios to obtain cell culture media containing the extract with extract fractions of 10%, 25%, 50%, and 100%.

[0060] Zn in the zinc alloy leachates of Example 1 and Comparative Example 1, before and after modification with selenium-containing polymers, was detected using inductively coupled plasma mass spectrometry (Agilent 7700 / 7800, USA). 2+ Content, results as follows Figure 4 As shown in Figure a, it can be seen that after 24 hours, the Zn content in the extract of the selenium-modified polymer group is significantly higher. 2+ The concentration was significantly lower than that of the unmodified extract, indicating that selenium-containing polymer modification could significantly alleviate Zn 2 +The burst release was observed, with the SePUA group showing a better mitigation effect. hBMMSCs cells were cultured in PM cell proliferation medium, cell proliferation medium containing different volume ratios of degradable zinc alloy extract (10%, 25%, 50%, and 100% Zn0.8Li as shown in the figure), and PM containing 10% DMSO. Cells were incubated in a 5% CO2, 37°C incubator, with the medium changed every 2–3 days. Cell viability was assessed using the CCK-8 assay kit (Beyotime, China) on days 0, 1, 3, 5, and 7. Results are shown in [Figure Number]. Figure 4 b. It can be seen that the cell culture media with 10% and 25% extract did not affect the proliferation of the cultured cells, while the cell proliferation level decreased significantly in the 50% and 100% extract concentration groups. Therefore, the cell culture medium with a 25% extract concentration was used in subsequent experiments.

[0061] Depend on Figure 4 As can be seen, when hBMMSCs cells were cultured in the cell proliferation medium containing 25% extract for 24 and 48 hours, the cell uptake of Se was significantly increased in the experimental group compared with the control group. This indicates that selenium can be effectively uptaken by cells after modification with selenium-containing polymers to exert subsequent effects.

[0062] The cell compatibility of the selenium-containing polymer-modified degradable zinc alloy prepared in Example 1 was evaluated using a live / dead cell staining kit (KGI Biotechnology, China). Human bone marrow mesenchymal stem cells (hBMMSCs) were seeded into well plates. On the second day, the medium was replaced with a 25% extract of the selenium-containing polymer-modified degradable zinc alloy at different hydrophilic group ratios and selenium concentrations. Cell viability was assessed on the third day using live / dead cell staining. Cells on the zinc plate surface were photographed using a TE2000-U inverted fluorescence microscope (Nikon, Japan) with F3.0 digital imaging system software (NIS-Elements, Japan). Live cells fluoresced in green, while dead cells fluoresced in red. Figure 4 As shown in d, there was no significant difference in the effect of 25% concentration culture medium prepared from zinc alloy extracts modified with selenium-containing polymers of different proportions and concentrations on cell viability.

[0063] Example 5: Detection of the osteogenic effect of in vitro mesenchymal stem cells on zinc alloy materials modified with selenium-containing polymers.

[0064] The in vitro mesenchymal stem cell-promoting effect of selenium-containing polymer-modified zinc alloy materials on osteoproliferation was evaluated using alkaline phosphatase (ALP) staining and quantification, alkaline phosphatase (ARS) staining and quantification, quantitative real-time PCR (qPCR), Western blotting, and immunofluorescence techniques.

[0065] Here, a 25% Se selenium-containing polymer with a hydrophilic group to selenium group ratio of 3:1 was selected. hBMMSCs cells were seeded in 24-well plates at a density of approximately 3000 cells / mL, with 0.5 mL per well. Cells were incubated with PM cell proliferation medium until 80% confluence was achieved, then replaced with the medium containing 25% selenium extract. The medium was changed every 2 days, and the cells were cultured at 37°C and 5% CO2 for 7 days. Afterward, the cells were washed three times with PBS, and staining and quantitative analysis were performed using an alkaline phosphatase (ALP) staining kit (Beyotime, China) and an alkaline phosphatase quantitative kit (Beyotime, China). The results are as follows: Figure 5 As shown in Figure a, the ALP staining of hBMMSCs cultured in SePUA-Zn0.8Li (Example 1) extract medium was generally deeper than that in the SePU-Zn0.8Li (Comparative Example 1) extract medium group, and the ALP activity was highest in the 25% Se 3 mg / mL group.

[0066] Figure 5 Figure b shows the ALP quantitative data of hBMMSCs cultured in SePUA-Zn0.8Li (Example 1) extract medium with different selenium group ratios and concentrations. The highest ALP activity was also observed in the 25% Se 3 mg / mL group.

[0067] The expression mechanism of osteogenic-related genes in hBMMSCs was analyzed using qPCR. Cells were seeded and cultured using the same methods as described above. When confluence reached 80%, the culture medium was changed to different 25% extract media. The extract material used in the control group was unmodified zinc alloy Zn0.8Li, while the experimental groups were selenium-containing polyurethane (Comparative Example 1) and selenium-containing polyurea (Example 1) modified with 25% Se groups at concentrations of 1, 3, and 5 mg / mL, respectively. These are abbreviated as 25-1SePU, 25-3SePU, 25-5SePU, 25-1SePUA, 25-3SePUA, and 25-5SePUA in the figure. The medium was changed every 48 hours. Samples were collected 7 days after induction, and the mRNA expression levels of two marker genes, osterix (OSX) and osteocalcin (OCN), were measured. The results are shown below. Figure 5 As shown in c. From Figure 5As can be seen, the expression levels of each marker gene in the degradable zinc alloy after surface modification with selenium-containing polymer compounds were upregulated compared with the unmodified group, and the expression levels of the marker gene in SePUA-Zn0.8Li in Example 1 were significantly higher than those in the control group SePU-Zn0.8Li.

[0068] The expression level of RUNX2 protein in hBMMSCs cultured in a biodegradable zinc alloy before and after modification with selenium-containing polymers was measured using Western blotting and immunofluorescence techniques. hBMMSCs were seeded in 24-well plates and incubated with proliferation medium until 80% confluence, then replaced with 25% cell culture medium containing extract. After 7 days of induction, the cells were centrifuged, and the supernatant was collected for analysis. The results are as follows: Figure 5 As shown in d and 5e. From Figure 5 As can be seen, the expression of osteogenic-associated protein RUNX2 increased in the degradable zinc alloy group modified with selenium-containing polymers (Example 1); Figure 5 As shown in e, the immunofluorescence results showed that the fluorescence intensity of RUNX2 and OCN in the selenium-containing polymer (Example 1) modified group was significantly stronger than that in the unmodified group, which is consistent with the immunofluorescence results, indicating that the selenium-containing polymer can promote osteogenic differentiation of hBMMSCs.

[0069] Example 6: Detection of in vitro macrophage polarization towards anti-inflammatory phenotype promoted by zinc alloy materials modified with selenium-containing polymers.

[0070] The effect of selenium-containing polymers modified with degradable zinc alloys on the polarization of THP-1-derived macrophages was evaluated. Human monocytic leukemia cells (THP-1) in suspension were centrifuged (600 rpm, 3 min), the supernatant was discarded, and the pellet was resuspended in cell proliferation medium (RPMI 1640, 10% FBS, 1% penicillin-streptomycin) to obtain a cell suspension. 100 ng / mL phorbol ester (PMA) was added. The cell suspension was seeded into well plates, and after 24 hours, adherent growth was induced under a microscope. The medium was then replaced with cell proliferation medium containing PMA extract. Samples were collected after 4 days, and qPCR was used to detect the expression levels of THP-1-derived macrophages related to pro-inflammatory and anti-inflammatory phenotypes. The results of selenium-containing polymers modified on the zinc alloy surface in Example 1 and Comparative Example 1 are shown below. Figure 6 As shown in a. After modification with all selenium-containing polymers, the expression levels of the pro-inflammatory gene iNOS were downregulated, and the expression levels of the anti-inflammatory gene ARG-1 were upregulated. Moreover, Example 1 showed a more significant regulatory effect than Comparative Example 1.

[0071] The levels of reactive oxygen species (ROS) in macrophages were measured (Solepro, China), and the results are as follows: Figure 6c shows that the fluorescence intensity of macrophages cultured in zinc alloy modified with selenium-containing polymers was significantly lower than that in the unmodified group, indicating that selenium-containing polymers can scavenge ROS generated in macrophages due to oxidative stress in the zinc alloy environment, thereby inhibiting the secretion of pro-inflammatory factors.

[0072] Figure 6 Western blotting and immunofluorescence studies at 6d and 6d used the 25% Se 3mg / mL SePUA-Zn0.8Li, which showed the best osteogenic effect, as the experimental group. These studies also demonstrated the regulatory effect of selenium-containing polymer modification on the expression levels of iNOS and ARG-1, indicating that selenium-containing polymers modifying degradable zinc alloys can better promote the polarization of human monocytes to an anti-inflammatory phenotype in vitro.

[0073] Example 7: In vivo osteopromoting effect of zinc alloy materials modified with selenium-containing polymer compounds.

[0074] Bilateral femoral defect modeling was performed on 6-8 week old male SD rats. The specific method was as follows: After anesthetizing the rats, a 1.5 mm diameter defect was created bilaterally on both femurs at a point 5 mm proximal to the lateral epicondyle, perpendicular to the long axis of the femur. Two types of biodegradable zinc alloy rods, one unmodified after grinding and the other modified with different selenium-containing polymers (25% Se 3 mg / mL), were implanted into the bone defect sites of the model rats. Three rats were in each group. At week 4 post-surgery, the rats were sacrificed, and the femurs were removed for Micro-CT scanning. The results are as follows: Figure 7 As shown in Figure a. In the unmodified group, there was no obvious high-density shadow around the implant, indicating almost no new bone formation. In Comparative Example 1, after the SePU polymer-modified zinc alloy rod was implanted, only a small amount of high-density shadow and new bone formation were observed around it. In Example 1, the SePUA polymer-modified group showed a large amount of high-density shadow around the implant, indicating more new bone formation than the SePU-modified group in Comparative Example 1. This suggests that the SePUA polymer-modified zinc alloy in Example 1 has a superior in vivo ossification effect, consistent with the in vitro results. Bone tissue sections and methylene blue-acid fuchsin staining were performed on the femurs of the unmodified zinc alloy group and the selenium-containing polymer-modified group in Example 1. The results are as follows. Figure 7 As shown in b, at 4 weeks, the unmodified zinc alloy group showed a small amount of discontinuous new bone formation, while the modified group of Example 1 formed more and more continuous new bone, which was more closely integrated with the implant material. At 8 weeks, although the bone volume in the unmodified zinc alloy group increased compared to 4 weeks, the new bone formation was still discontinuous and fragmented, while the modified group of Example 1 formed more and more continuous new bone, which was more tightly integrated with the implant. These results indicate that modification with selenium-containing polymer compounds enhances the osseointegration capacity of metallic materials, and that modifying the surface of biodegradable zinc alloy with selenium-containing polyurea in Example 1 has a better osteogenic effect than modifying the selenium-containing polyurethane in Comparative Example 1, thus enhancing bone regeneration around the metallic material.

[0075] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations. The above embodiments or implementations are merely illustrative examples of this invention, and it can also be implemented in other specific ways or forms without departing from its gist or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of this invention should be defined by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of this invention.

Claims

1. A zinc alloy material modified with a selenium-containing polymer compound, characterized in that: The zinc alloy material modified with the selenium-containing polymer compound is obtained by coordination compounding of the selenium-containing polymer compound with zinc alloy. The selenium-containing polymer compound is prepared by reaction from raw materials containing H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate, and polyethylene glycol monomethyl ether.

2. The zinc alloy material modified with selenium-containing polymer compounds according to claim 1, characterized in that: The zinc alloy is selected from zinc-lithium alloys and / or zinc-lithium-magnesium alloys.

3. The method for preparing zinc alloy materials modified with selenium-containing polymer compounds according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Preparation of selenium-containing polymers: H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate, polyethylene glycol monomethyl ether and catalyst are mixed in an organic solvent and reacted. The product obtained from the reaction is recrystallized and washed. (2) Dissolve the selenium-containing polymer compound obtained in step (1) in an organic solvent, and then soak it in a zinc alloy for modification reaction.

4. The preparation method according to claim 3, characterized in that: In step (1), The organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate, and n-butyl acetate; The diisocyanate is selected from one or more of toluene 2,4-diisocyanate, hexamethylene diisocyanate, diphenylmethane diisocyanate, and isophorone diisocyanate; The molar ratio of H2N-(CH2)3-Se-(CH2)3-NH2, 1,4-bis(2-hydroxyethyl)piperazine, diisocyanate, and polyethylene glycol monomethyl ether is x:1:(1.1x+1.1):(0.2x+0.2), where x is 0.1 to 10; The catalyst is dibutyltin dilaurate.

5. The preparation method according to claim 3, characterized in that: In step (1), the reaction temperature is 30-100℃ and the reaction time is 1-48 hours.

6. The preparation method according to claim 3, characterized in that: In step (1), the recrystallization is carried out using diethyl ether, and the washing is carried out using ethanol with a volume percentage concentration of 5-50%.

7. The preparation method according to claim 3, characterized in that: In step (2), The organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide; The concentration of selenium-containing polymers dissolved in organic solvents is 1-10 mg / mL.

8. The preparation method according to claim 3, characterized in that: In step (2), The zinc alloy is a zinc alloy sheet or a zinc alloy rod; The zinc alloy is polished and ultrasonically cleaned before use.

9. The preparation method according to claim 3, characterized in that: In step (2), the modification reaction takes 1-3 hours.

10. The use of the zinc alloy material modified with selenium-containing polymer compounds according to claim 1 or 2 for the preparation of dental implants or orthopedic implants.

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