CN116005053A - Mg-Sr-Zn magnesium alloy with bone immunoregulation function and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Mg-Sr-Zn series magnesium alloy with bone immune regulation function, a preparation method and application thereof, and belongs to the field of medical magnesium alloy materials. The Mg-Sr-Zn-based magnesium alloy of the present invention is composed of Mg, Sr and Zn; based on the mass of the Mg-Sr-Zn-based magnesium alloy, the mass percentage of the Sr is 1%; the Zn The mass percentage is 2%; the balance is Mg. The Mg-Sr-Zn series magnesium alloy provided by the present invention has good biocompatibility and corrosion resistance, meets the requirements of mechanical properties, and can regulate the expression of immune osteogenesis-related cytokines such as TNF-α, IL10, VEGF, etc. It can promote the function of bone regeneration through the bone immune regulation mechanism, and can be used as a medical implant material at the same time.

Description

A Mg-Sr-Zn series magnesium alloy with bone immune regulation function and its preparation method and application

technical field

The invention relates to the field of medical magnesium alloy materials, in particular to a Mg-Sr-Zn series magnesium alloy with bone immune regulation function and its preparation method and application.

Background technique

For a long time, traditional orthopedic implant materials represented by stainless steel, cobalt-chromium-based alloys, titanium and titanium alloys have been widely used clinically. However, in long-term clinical practice, it has been found that these traditional orthopedic implants have relatively stable chemical properties, and they mainly play the role of mechanical fixation and support after implantation in the human body, and can promote the healing of affected tissue and the formation of new bone. limited. At the same time, these traditional orthopedic implants either remain in the body for a long time after being implanted in the body, or are removed through a second operation after the affected tissue heals. Long-term retention of traditional orthopedic implants in the body will lead to a series of clinical problems such as rejection, loosening of implants, and interference with MRI/CT imaging examinations. Secondary surgical removal not only increases the pain and medical burden of patients, but also It also wastes a lot of medical resources. It is of great significance to develop and design orthopedic implant materials with in vivo degradability. Polymer implants such as PLA and PLGA are biodegradable, but limited by the inherent properties of polymers, the implant has low strength and poor mechanical properties, and it is often difficult to provide mechanical support in orthopedic implants role. At the same time, its degradation process will make the surrounding of the implant appear acidic, which will lead to the occurrence of aseptic inflammation. In recent years, with the continuous development of degradable implant materials, biomedical metal degradable orthopedic implant materials represented by magnesium and its alloys have attracted more and more attention.

In the early research of biomedical degradable magnesium alloys, people usually use WE43, AZ31, AZ91 and other industrially applied magnesium alloys to evaluate the material properties and biocompatibility of magnesium alloys, and there is no application scenario for human bone tissue. The types and contents of alloying elements in magnesium alloys were optimized. The content of rare earth elements in the normal human body is extremely low. At present, its metabolic pathway in the body is not very clear. Excessive accumulation of rare earth elements in the body is considered to have certain toxic and side effects. The accumulation of Al element in the body is also considered to be related to Alzheimer's disease and Parkinson's disease, and Al element is also considered to be neurotoxic. Therefore, biological safety is the primary consideration in the design of medical degradable magnesium alloys.

As a foreign body, medical degradable magnesium alloy implants are implanted into the human body and come into contact with human tissues and body fluids. Proteins in blood and body fluids will quickly adhere to the surface of the implants, and then platelets are activated to form blood clots. After that, monocytes and other inflammatory cells will be recruited at the implant-bone tissue interface and differentiate into macrophages, causing acute inflammation and other immune responses, while secreting a large number of cytokines to participate in subsequent related regulation. But for now, people's research on medical degradable magnesium alloys is mainly focused on the evaluation of the osteogenic function of magnesium alloy implants in vivo, especially for the regulation of magnesium alloy implants on surrounding tissues. Immunomodulatory effects and their possible osteogenic mechanisms are still poorly understood. In recent years, it has been found that the immune system shares some cytokines, receptors, signaling molecules and transcription factors with the skeletal system, and the immune system may also play an important role in bone metabolism, which also led to the concept of bone immune regulation, which also It suggests that we can make full use of bone immune regulation, design a degradable magnesium alloy implant with appropriate immune regulation function, promote new bone formation, and accelerate bone healing.

Contents of the invention

The purpose of the present invention is to provide a Mg-Sr-Zn series magnesium alloy with bone immune regulation function and its preparation method and application. The Mg-Sr-Zn series magnesium alloy provided by the present invention has good biocompatibility and resistance Corrosion performance, meet the requirements of mechanical properties, and can regulate the expression of immune osteogenesis-related cytokines such as TNF-α, IL10, VEGF, etc., can promote bone regeneration through the bone immune regulation mechanism, and can be used as a medical implant material.

The present invention firstly provides a Mg-Sr-Zn series magnesium alloy, the Mg-Sr-Zn series magnesium alloy is composed of Mg, Sr and Zn;

Based on the mass of the Mg-Sr-Zn series magnesium alloy, the mass percentage of the Sr is 1%; the mass percentage of the Zn is 2%; the balance is Mg.

In the above Mg-Sr-Zn series magnesium alloy, the surface of the Mg-Sr-Zn series magnesium alloy is coated with a degradable ceramic coating.

The material of the degradable ceramic coating can specifically be one of hydroxyapatite, strontium-containing hydroxyapatite, fluorine-containing hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate and tetracalcium oxyphosphate any combination of one or more;

The thickness of the degradable ceramic coating is 0.01-5mm;

The degradable ceramic coating can be applied by plasma spraying, electrodeposition or micro-arc oxidation methods.

The Mg-Sr-Zn series magnesium alloy is an extruded alloy.

There are some unavoidable impurities in the above-mentioned Mg-Sr-Zn series magnesium alloy, wherein the impurity elements and their contents are respectively: iron (Fe)≤0.1wt%, copper (Cu)≤0.1wt%, nickel (Ni)≤ 0.1 wt%.

The present invention also provides a preparation method of the above-mentioned Mg-Sr-Zn series magnesium alloy, comprising the following steps:

The Mg, Sr and Zn are mixed to obtain a mixture, the mixture is melted under the protection of an inert atmosphere, and the Mg-Sr-Zn series magnesium alloy is obtained after cooling.

The above preparation method also includes the step of machining the Mg-Sr-Zn series magnesium alloy; the machining includes rolling and/or forging and/or extrusion and/or rapid solidification.

In the above preparation method, the extrusion temperature range of the extrusion step is 200-400° C., the extrusion speed is 0.1-30 m/min, and the extrusion ratio is 10-100.

Specifically, the extrusion temperature in the extrusion step is 320° C., the extrusion speed is 0.12 m/min, and the extrusion ratio is 17.36.

In the above preparation method, the inert atmosphere is an argon atmosphere.

In the above preparation method, the melting temperature is 650-850°C; specifically, it may be 670°C.

Finally, the present invention provides the application of the above-mentioned Mg-Sr-Zn series magnesium alloy in the preparation of medical implants;

The Mg-Sr-Zn series magnesium alloy can regulate the secretion and expression of immune osteogenesis-related factors, thereby promoting new bone formation and accelerating bone tissue healing.

In order to slow down the degradation speed of the Mg-Sr-Zn series magnesium alloy medical implant, the surface of the medical implant can also be coated with a degradable polymer coating or a degradable ceramic coating.

The medical implant can be used as a therapeutic implant stent, a bone repair device or a dental repair device; the implant stent can be a vascular stent, an esophageal stent, an intestinal stent, a tracheal stent, a biliary stent or a urethral stent; The above-mentioned bone repair device can be a bone tissue repair bracket, a bone set, a fixation wire, a fixation screw, a fixation rivet, a fixation pin, a bone splint, an intramedullary nail or a bone sleeve, etc.

The present invention has the following advantages:

The Mg-Sr-Zn series magnesium alloy of the present invention selects essential metal elements for the human body, does not contain harmful or potentially harmful elements, and the alloying elements Sr and Zn in it are metal elements necessary for the human body, directly participate in bone metabolism, and can promote osteogenesis Regeneration, inhibition of bone resorption. Zn ²⁺ , Sr ²⁺ plasma will be released locally along with the gradual degradation of the Mg-Sr-Zn magnesium alloy implant in the body, and these alloy element ions released into the surrounding tissues such as Zn ²⁺ , Sr ²⁺ can be regulated The secretion of immune osteogenesis-related factors such as TNF-α, IL10, and VEGF can regulate the formation of new bone in the affected area through the bone immune regulation mechanism and accelerate the healing of bone tissue in the affected area.

The method of the present invention realizes regulating and controlling the mechanical properties and degradation speed, mechanical properties and corrosion resistance of medical implants through the cooperation of composition design and preparation process (such as extrusion deformation), wherein the Mg-1Sr-2Zn alloy implant has Optimal application prospects.

The Mg-Sr-Zn series magnesium alloy of the present invention has no toxic effect on the human body, has good histocompatibility, mechanical properties and adjustable degradation rate, and is suitable for the field of degradable orthopedic implant instruments.

Description of drawings

FIG. 1 is a metallographic microstructure diagram of the extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy prepared in Example 1.

Fig. 2 is the room temperature tensile property curve of the extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy prepared in Example 1.

Fig. 3 is the electrochemical performance of the extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy prepared in Example 1 in Hank's solution.

Fig. 4 is the surface morphology of the extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy prepared in Example 1 after soaking in Hank's solution.

Fig. 5 is the concentration of alloy element ions in the solution after soaking the extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy prepared in Example 1 in Hank's solution.

Fig. 6 is the cytotoxicity diagram of the extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2 alloy prepared in Example 1 to hBMSCs cells.

Figure 7 shows the alkaline phosphatase activity of hBMSCs cells cultured in the extract solution prepared in Example 6 for different days.

Figure 8 is the quantitative results of Alizarin Red staining of hBMSCs cells cultured in the extract solution prepared in Example 7 for different days.

Figure 9 shows the results of secretion of inflammatory factors after the extruded Mg-1Sr-2Zn alloy prepared in Example 1 was subcutaneously implanted in SD rats for 3 days.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, preparation extruded state Mg-Sr-Zn alloy

The test raw materials are pure Mg (99.7wt.%), Sr powder (99.9%) and Zn powder (99.5wt.%) according to the nominal composition Mg-xSr-yZn (x=0.2,0.5,1,2; y=2, 4, 6) proportioning, under 670 ℃ temperature, smelting under the protective atmosphere of Ar gas (99.99%, volume fraction), heat preservation for 40 minutes after the experimental material is fully melted, adopt the mode of precision casting subsequently (pouring temperature is 680～ 700°C, the mold temperature is 250°C) and the molten material is poured into a pre-designed mold, and the as-cast Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) Alloy ingots, process Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy ingots into Φ39.6mm×50mm ingots, solidify at 340°C Dissolve for 4 hours. The ingot was preheated to 340°C and then held for 20 minutes. The temperature of the mold and extrusion barrel was both 295°C, the extrusion temperature was 320°C, and the extrusion speed was 2mm/s.

In this example, an extruded Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloy was obtained, and its microstructure is shown in FIG. 1 . It can be seen from Fig. 1 that all Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloys present typical metallographic morphology of extruded alloys, and the grain size is relatively small. Small, the refinement is obvious, and at the same time, with the increase of Zn content in the alloy, the deposition of the second phase in the alloy gradually increases.

Among them, the above-mentioned x=0.2, 0.5, 1, 2 means that the mass of Sr accounts for 0.2%, 0.5%, 1% or 2% of the alloy mass; y = 2, 4, 6 means that the mass of Zn accounts for the mass of the alloy 2%, 4% or 6% of the

Example 2, room temperature tensile properties of extruded Mg-xSr-yZn (x=0.2,0.5,1,2; y=2,4,6) alloy

The extruded Mg-xSr-yZn (x=0.2,0.5,1,2; y=2,4,6) alloy prepared in Example 1 was prepared according to the ASTM-E8-04 tensile test standard to prepare tensile samples, SiC The sandpaper is polished to 2000#, and the tensile test is carried out at room temperature with a general material tensile testing machine, and the tensile speed is 0.5mm/min.

The tensile properties at room temperature of the Mg-xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) alloys prepared by the present invention are shown in FIG. 2 . It can be seen from Figure 2 that when the Sr content is the same, the Zn content has little effect on the yield strength and tensile strength of the alloy, but has a greater influence on the elongation of the alloy, and as the Sr content increases to 2%, the yield strength and tensile strength of the alloy The tensile strength has been improved to a certain extent. Except for the alloy with 0.5% Sr content, the elongation of the alloy decreases with the increase of Zn content in other alloys.

Embodiment 3, the electrochemical performance of extruded state Mg-xSr-yZn (x=0.2,0.5,1,2; y=2,4,6) alloy

The Mg-xSr-yZn (x = 0.2, 0.5, 1, 2; y = 2, 4, 6) alloy wire cutting process prepared in Example 1 was processed into a block sample of Φ10mm × 2mm, polished to 2000 with sandpaper #. Electrochemical tests were then performed in Hank's simulated body fluid at 37±0.5°C.

The corrosion potential-corrosion current curve of the Mg-xSr-yZn (x=0.2,0.5,1,2; y=2,4,6) alloy prepared by the present invention is as shown in Figure 3, and its corrosion current density, open circuit potential and The self-corrosion potential is shown in Table 1. Corrosion current density, self-corrosion potential, open circuit of Mg--xSr-yZn (x=0.2, 0.5, 1, 2; y=2, 4, 6) extruded alloy prepared in Example 1 in Table 1 in Hank's solution potential.

From the data in Figure 3 and Table 1, it can be seen that when the Sr content is constant, the corrosion current density and corrosion rate of the alloy increase and decrease with the increase and decrease of the Zn content, indicating that the addition of Zn content can improve the corrosion resistance of the Mg-Sr-Zn alloy .

Example 4

Adopt the method in the embodiment 3 to prepare the bulk test sample of Φ 10mm * 2mm, according to the ratio of surface area/Hank's volume is 1/40cm ² ·mL ^-1 soak 3 pieces of samples in the Hank's solution of 37 ± 0.5 ℃, soak 3 The surface morphology of the material after 1 day is shown in Fig. 4. It can be seen from Figure 4 that after soaking for 3 days, the surface of all alloys is covered with a layer of corrosion products, and with the increase of Zn content, the content and thickness of surface corrosion products increase, which can also reduce the corrosion rate of the alloy. After soaking for 10 days, the concentration of each ion in the solution is shown in Figure 5. It can be seen from Figure 5 that after soaking, the solution is mainly composed of Mg ions, and its content is significantly higher than that of Zn ions and Sr ions. When the Sr content is 2%, the concentration of alloy element ions in the solution is higher than that of other alloys.

Example 5

Adopt the method in embodiment 3 to prepare the bulk test sample of Φ 10mm * 2mm, according to the surface area/leaching solution volume ratio after 4h of ultraviolet radiation sterilization, be 1.25cm ² ·mL ^-1 standard preparation leaching solution (after sterilizing The sample was soaked in serum-free MEM medium, after 72 hours in the incubator (37°C, 5% CO ₂ ), the medium was taken out and centrifuged, and the obtained supernatant was the extract) and used Human bone marrow mesenchymal stem cells (hBMSCs) evaluated the cytotoxicity of the Mg-xSr-2Zn (x=0.2, 0.5, 1, 2) alloy obtained in the present invention. Figure 6 shows the cell viability after the cells were cultured in the extract solution for 2d, 4d and 7d under the conditions of 37°C and 5% CO ₂ respectively. It can be seen from Figure 6 that as the culture time increased from 2 days to 7 days, the proliferation rate of hBMSCs cells in the alloy leaching solution gradually increased. In different alloys, the cell survival rate was not significantly different, indicating that Mg-Sr- Zn alloy has good cell compatibility.

Example 6

After the hBMSCs cells cover 80% of the culture flask, digest the cells with 0.25wt% trypsin (manufacturer: Hyclone; product number: SH30042.01), and resuspend the collected cells in MEM to a cell concentration of 1× ¹⁰⁴ The cell suspension of /mL is inoculated in the 96-well plate, 100 μ L/ hole, and after 24h, after the cells adhere to the wall completely, replace the culture medium with the extract of the experimental material (the preparation method of the extract is the same as that of Example 5, and add Add induction solution to it, and the final concentration is as follows: 10mmol/L beta-glycerophosphate sodium, 50ug/mL ascorbic acid, 10nmol/L dexamethasone, both are Sigma), respectively at 37°C, 5% CO Under the condition _of 7d, 14d, Replace the fresh extraction solution every 2 days, discard the old medium, wash with PBS solution 3 times, add 200 μL 0.1wt% Triton X-100 to each well, and lyse overnight at 4°C for later use. Intracellular alkaline phosphatase (ALP) activity was detected by p-NPP (Sigma). The specific method is as follows: Take 50 μL of cell lysate, add 50 μL of ALP substrate reaction solution, keep the water bath at 37°C for 30 minutes, add 50 μL of 0.1mol/L NaOH solution The reaction was terminated, and the absorbance value was measured at a wavelength of 405 nm in a microplate reader. The total intracellular protein concentration (mg/L) was measured by Pierce BCA kit, and the total protein amount was calculated according to the volume (50 μL) and the total protein concentration. The alkaline phosphatase activity of the cells is shown in FIG. 7 . It can be seen from Figure 7 that as the culture time increased from 7 days to 14 days, the ALP activity of hBMSCs cells increased significantly, and the ALP activity of hBMSCs cells in the alloy extract was significantly higher than that of the blank control group, indicating that the Mg-Sr-Zn alloy can promote hBMSCs osteogenic function.

Example 7

After the hBMSCs cells cover 80% of the culture flask, digest the cells with 0.25% trypsin, resuspend the collected cells in MEM to a cell suspension with a cell concentration of 5× ¹⁰ cells/mL and inoculate them into a 24-well plate After 24 hours, after the cells have adhered to the wall completely, replace the culture medium with the extract of the experimental material (the preparation method of the extract is the same as in Example 5, and the induction solution is added thereto, and the final concentration is as follows: 10mmol/L β-sodium glycerophosphate , 50ug/mL ascorbic acid, 10nmol/L dexamethasone, both are Sigma), cultured at 37°C, 5% CO ₂ for 7d, 14d and 21d, respectively, changing the extract every other day, discarding the old medium, PBS Wash 3 times and fix with 4% (volume fraction) paraformaldehyde. Alizarin red staining kit was used for calcium nodule staining. After staining, excess alizarin red staining solution was washed with PBS. Calcium nodules were dissolved with 10% (mass fraction) cetylpyridinium chloride, and the absorbance value was measured at a wavelength of 562 nm in a microplate reader. Alizarin red staining absorbance values of hBMSCs cells are shown in Figure 8. It can be seen from Figure 8 that as the culture time increased from 7 days to 21 days, the calcium nodule deposition of hBMSCs cells continued to increase, and the Mg-1Sr-2Zn alloy showed the best mineralization ability. Compared with the blank control group, the mineralization ability of the Mg-Sr-Zn alloy group is stronger, indicating that it can promote mineralization in vitro.

Example 8

The method in Example 4 was used to prepare a block test sample of Φ10 mm×2 mm, sterilized by ultraviolet irradiation for 4 hours, and then implanted into the peritoneal cavity of SD rats subcutaneously. The implant was taken out 3 days after implantation, and 3 mL of sterile saline was used to wash the implant site. After washing three times, it was collected in a sterile centrifuge tube, and then ELISA kit was used to detect VEGF, TNF-α, The content of immune-related cytokines such as IL-10. The contents of different factors are shown in Figure 9. It can be seen from Figure 9 that, compared with the pure titanium control, the Mg-1Sr-2Zn alloy (Mg in Figure 9 is the Mg-1Sr-2Zn alloy group) can significantly increase the expression of TNF-α after implantation, while significantly reducing VEGF and The expression of IL-10 also indicates that the Mg-1Sr-2Zn alloy can regulate the expression of immune osteogenesis-related cytokines in the early stage of implantation.

Claims (9)

1. A Mg-Sr-Zn series magnesium alloy, characterized in that: the Mg-Sr-Zn series magnesium alloy is composed of Mg, Sr and Zn; Based on the mass of the Mg-Sr-Zn series magnesium alloy, the mass percentage of the Sr is 1%; the mass percentage of the Zn is 2%; the balance is Mg. 2. The Mg-Sr-Zn series magnesium alloy according to claim 1, characterized in that: the surface of the Mg-Sr-Zn series magnesium alloy is coated with a degradable ceramic coating. 3. The Mg-Sr-Zn series magnesium alloy according to claim 2, characterized in that: the material of the degradable ceramic coating is hydroxyapatite, strontium-containing hydroxyapatite, fluorine-containing hydroxyapatite , any combination of one or more of α-tricalcium phosphate, β-tricalcium phosphate and oxytetracalcium phosphate; The thickness of the degradable ceramic coating is 0.01-5mm; The degradable ceramic coating is applied by plasma spraying, electrodeposition or micro-arc oxidation. 4. The Mg-Sr-Zn-based magnesium alloy according to any one of claims 1-3, characterized in that: the Mg-Sr-Zn-based magnesium alloy is an extruded alloy. 5. the preparation method of the Mg-Sr-Zn series magnesium alloy described in any one in claim 1-4, comprises the steps: The Mg, Sr and Zn are mixed to obtain a mixture, the mixture is melted under the protection of an inert atmosphere, and the Mg-Sr-Zn series magnesium alloy is obtained after cooling. 6. The preparation method according to claim 5, characterized in that: the method also includes the step of machining the Mg-Sr-Zn series magnesium alloy; the machining includes rolling and/or forging and /or extrusion and/or rapid setting. 7. The preparation method according to claim 5 or 6, characterized in that: the inert atmosphere is an argon atmosphere. 8. The preparation method according to any one of claims 5-7, characterized in that: the melting temperature is 700-850°C. 9. The application of the Mg-Sr-Zn series magnesium alloy described in any one of claims 1-4 in the preparation of medical implants; The Mg-Sr-Zn series magnesium alloy can regulate the secretion and expression of immune osteogenesis-related factors, thereby promoting new bone formation and accelerating bone tissue healing.

Images