CN114984306B - Method for constructing biological protein mineralized coating on surface of Zr-based alloy - Google Patents

Method for constructing biological protein mineralized coating on surface of Zr-based alloy Download PDF

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CN114984306B
CN114984306B CN202210704820.3A CN202210704820A CN114984306B CN 114984306 B CN114984306 B CN 114984306B CN 202210704820 A CN202210704820 A CN 202210704820A CN 114984306 B CN114984306 B CN 114984306B
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刘立斌
薛人豪
章立钢
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Central South University
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Abstract

The invention relates to the technical field of biomedical materials, and provides a method for constructing a bioprotein mineralized coating on the surface of a Zr-based alloy. The invention adopts elastin and cross-linking agent as raw materials, forms a protein film on the surface of Zr-based alloy by a protein molecule self-assembly method, and then induces the film to mineralize in a mineralized liquid environment, so as to realize the construction of a biological protein mineralized coating on the surface of the Zr-based alloy. The method provided by the invention successfully constructs the mineralized coating on the surface of the Zr-based alloy, and the construction process has simple steps and is easy to operate, and has obvious advantages compared with other complex surface treatment technologies. Furthermore, the invention adopts Zr-xNb-yTi (x = 8-16, y = 0-16) alloy as the substrate, the Zr-based alloy has low elastic modulus, low magnetization rate and better biocompatibility, and after the mineralized coating is constructed, the biocompatibility of the Zr-based alloy is greatly improved, and the Zr-xNb-yTi alloy has larger potential clinical use value.

Description

Method for constructing biological protein mineralized coating on surface of Zr-based alloy
The present application claims priority from the chinese patent application entitled "a method for constructing a bioprotein mineralization coating on a surface of a Zr-based alloy" filed by the chinese patent office at 2022, 03, 17.d., 202210264686.X, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a method for constructing a bioprotein mineralized coating on the surface of a Zr-based alloy.
Background
Biomedical materials are functional materials used in clinical treatments, repair or replacement of human tissues, organs or to enhance their function. General requirements for biomedical materials include: (1) Must meet the regulation of relevant standard, and should have no toxicity, no pyrogen reaction, no teratogenesis, no carcinogenesis, no anaphylaxis, no interference to the immune mechanism of organism, good blood compatibility and histocompatibility, etc. And (2) good biological stability. For biomedical materials to be implanted in the body for a long period of time, the structural properties of the material must be stable. And (3) has certain biological activity. As an implant material, biomedical materials need to ensure that the materials and biological tissues do not have any adverse physiochemical reaction, and the implant material needs to have a certain degree of biological activity, induce osseointegration and accelerate bone regeneration on the premise of not having cytotoxicity, causing the problems of distortion and the like. (4) excellent corrosion resistance: the implant material exists in a living body for a long time, and is exposed in body fluid for a long time, the temperature of the body fluid is about 37 ℃, and the body fluid contains K, na, cl and other ions and has certain corrosiveness. Once the material is corroded in the service process, the function of the material itself may be weakened or lost, and even certain damage may be caused to the organism, so that the material is required to have excellent corrosion resistance. (5) The material has high strength, and can be subjected to the action of external load in the service process, so that the material is required to have high strength in order to ensure the stable work of the material. (6) lower modulus of elasticity. The elastic modulus of bones is mostly in the range of 10-30 GPa, and if the elastic modulus of the implant material is far higher than that of bones, the stress born by the implant is too large, so that the human bones are hardly stressed, and the phenomenon of stress shielding is caused. (7) lower magnetic susceptibility. The metal-based biomaterial has too high magnetic susceptibility, generates 'artifacts' in the MRI diagnosis process, seriously influences the judgment of doctors, and causes the problems of fever and the like which endanger the safety of patients due to the too high magnetic susceptibility.
Excellent biocompatibility is one of the key factors of biomaterials and also one of the important factors hindering the development of biomaterials. Many metal materials with excellent mechanical properties contain sensitizing and even cytotoxic alloying elements, which greatly affect the clinical application value of the metal materials.
Biomineralization refers to the process of generating inorganic minerals under the control of different biomacromolecules under strict biological control conditions, and tissues with the structure have different functions. The elastic self-assembly (ELRs) technology is a technology that an elastin film with different biological epitopes is attached to the surface of a specified material, and a mineralized coating is formed by crystallization in the elastin film through a specific regulation technology.
At present, the self-assembly ELRs technology is mainly applied to substrates such as high polymers, silicon wafers and the like, the surface chemical properties of a metal substrate at normal temperature and low temperature are very stable, and the corrosion resistance and the stability of metal surfaces with different components are different, so that the surface modification difficulty is large, the research on modifying the surface of the metal substrate is less, and no related report of constructing a mineralized coating on the metal substrate by adopting an elastin self-assembly method exists at present.
Disclosure of Invention
In view of the above, the present invention provides a method for constructing a bio-protein mineralized coating on the surface of a Zr-based alloy. The method provided by the invention can construct the biological protein mineralized coating on the surface of the Zr-based alloy, and improve the biocompatibility of the Zr-based alloy.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for constructing a biological protein mineralized coating on the surface of a Zr-based alloy comprises the following steps:
(1) Soaking the Zr-based alloy in a protein solution to form a protein film on the surface of the Zr-based alloy; the protein solution comprises the components of elastin, a cross-linking agent and a solvent;
(2) Immersing the Zr-based alloy with the formed protein film into a mineralization liquid for mineralization reaction, and forming a biological protein mineralization coating on the surface of the Zr-based alloy; the components of the mineralized liquid comprise sodium fluoride, hydroxyapatite and deionized water.
Preferably, the sequence of the elastin is MESLLP- [ ((VPGIG) 2 VPGKG(VPGIG) 2 ) 2 -DDDEEKFLRRIGRFG-((VPGIG) 2 VPGKG(VPGIG) 2 ) 2 ] 3 -V; the concentration of the elastin in the protein solution is 1-100 mg/mL.
Preferably, the cross-linking agent is hexamethylene diisocyanate, and the molar ratio of the cross-linking agent to the elastin is (3-150): 1.
Preferably, the solvent in the protein solution is a mixed solvent of dimethyl sulfoxide and dimethylformamide, and the volume ratio of the dimethyl sulfoxide to the dimethylformamide in the mixed solvent is 1 (6-10).
Preferably, the soaking in the step (1) is carried out at room temperature for 8-24 h under the standing condition; the relative humidity of the environment during the soaking process is below 20%.
Preferably, the concentration of sodium fluoride in the mineralized liquid is 1-5 mmol/L, the concentration of hydroxyapatite is 1-5 mmol/L, and the pH value of the mineralized liquid is 5.5-6.5.
Preferably, the temperature of the mineralization reaction is 35-39 ℃, and the time is 6-10 days.
Preferably, the Zr-based alloy comprises the following components in percentage by mass: 8 to 16 percent of Nb, 0 to 16 percent of Ti, and the balance of zirconium and inevitable impurities.
The invention also provides a Zr-based biomedical material, which comprises the Zr-based alloy and the bioprotein mineralization coating attached to the surface of the Zr-based alloy, wherein the bioprotein mineralization coating is constructed by the method in the scheme.
The invention provides a method for constructing a biological protein mineralized coating on the surface of a Zr-based alloy, which comprises the following steps: (1) Soaking the Zr-based alloy in a protein solution to form a protein film on the surface of the Zr-based alloy; the protein solution comprises the components of elastin, cross-linking agent and solvent; (2) Immersing the Zr-based alloy with the formed protein film into a mineralization liquid for mineralization reaction, and forming a biological protein mineralization coating on the surface of the Zr-based alloy; the components of the mineralized liquid comprise sodium fluoride, hydroxyapatite and deionized water. The invention adopts elastin and cross-linking agent as raw materials, forms protein film on the surface of Zr-based alloy by protein molecule self-assembly method, then induces film mineralization in specific mineralized liquid environment, realizes the construction of biological protein mineralized coating on the surface of Zr-based alloy. The method successfully constructs the mineralized coating on the surface of the Zr-based alloy, has simple construction process and easy operation, and has obvious advantages compared with other complex surface treatment technologies.
Furthermore, the Zr-xNb-yTi (x = 8-16, y = 0-16) alloy is used as a substrate, the Zr-based alloy is an ideal biological metal material, has low elastic modulus, low magnetization rate and good biocompatibility, and after the mineralized coating is constructed, the biocompatibility of the Zr-based alloy is greatly improved, has certain bioactivity, and improves the potential clinical use value of the metal medical material.
Drawings
FIG. 1 is an SEM picture of an unmineralized Zr-based alloy surface;
FIG. 2 is an SEM picture of the surface of the mineralized Zr-based alloy in example 1;
FIG. 3 is an XRD pattern of the surface of the mineralized Zr-based alloy in example 1;
fig. 4 is an SEM picture of osteoblasts on the surface of the mineralized Zr-based alloy.
Detailed Description
The invention provides a method for constructing a biological protein mineralized coating on the surface of a Zr-based alloy, which comprises the following steps:
(1) Soaking the Zr-based alloy in a protein solution to form a protein film on the surface of the Zr-based alloy; the protein solution comprises the components of elastin, a cross-linking agent and a solvent;
(2) Immersing the Zr-based alloy with the formed protein film into a mineralization liquid for mineralization reaction, and forming a biological protein mineralization coating on the surface of the Zr-based alloy; the components of the mineralized liquid comprise sodium fluoride, hydroxyapatite and deionized water.
The Zr-based alloy is soaked in the protein solution, and a protein film is formed on the surface of the Zr-based alloy. In the invention, the Zr-based alloy comprises the following components in percentage by mass: 8-16% of Nb, 0-16% of Ti and the balance of zirconium and inevitable impurities, wherein the mass fraction of Nb is preferably 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or 16%, and the mass fraction of Ti is preferably 0%, 4%, 8%, 12% or 16%. In the present invention, the Zr-based alloy is represented by Zr-xNb-yTi (x =8 to 16, y =0 to 16), and the Zr-based alloy is specifically Zr-16Nb, zr-16Nb-4Ti, zr-16Nb-8Ti, zr-16Nb-12Ti, zr-16Nb-16Ti, zr-8Nb-12Ti or Zr-12Nb-8Ti depending on the Ti content. The Zr-based alloy with the components has lower elastic modulus and low magnetic susceptibility, can effectively reduce the influence of stress shielding and artifact on clinical diagnosis, has excellent biocompatibility for alloying elements and matrix elements, and is an ideal biological metal material.
In the present invention, the components of the protein solution include elastin (ELR protein), a cross-linking agent and a solvent, the sequence of the elastin preferably being MESLLP- [ ((VPGIG) 2 VPGKG(VPGIG) 2 ) 2 -DDDEEKFLRRIGRFG-((VPGIG) 2 VPGKG(VPGIG) 2 ) 2 ] 3 -V; the invention preferably adopts the elastin with the sequence, has excellent mineralization effect, and is flexible and can be modified; in the present invention, the purity of the elastin is preferably greater than 95%, and the concentration of elastin in the protein solution is preferably 1-100 mg/mL, more preferably 1-60 mg/mL, even more preferably 1.5-20 mg/mL, and even more preferably 5-20 mg/mL; the source of the elastin in the present invention is not particularly required and commercially available products well known to those skilled in the art may be used. In the present invention, the crosslinking agent is preferably hexamethylene diiso-etherCyanate ester (HDI), the molar ratio of said cross-linking agent to said elastin being preferably (3-150): 1, more preferably (5-60): 1; the solvent in the protein solution is a mixed solvent of dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF), the volume ratio of dimethyl sulfoxide to dimethylformamide in the mixed solvent is preferably 1 (6-10), more preferably 1 (8.5-9.5), and more preferably 1:9. In the present invention, the protein solution is preferably prepared by: firstly, dissolving elastin in a solvent, and then adding a cross-linking agent to obtain the protein solution; the formulation of the protein solution is preferably carried out in a glove box, the relative humidity in which is preferably below 20%.
In the invention, the soaking temperature is preferably room temperature, the time is preferably 8-24 h, and more preferably 12-16 h, and the soaking is preferably carried out under a standing condition; the relative humidity of the environment in the soaking process is preferably below 20%; in a particular embodiment of the invention, the soaking is preferably performed in a glove box. During the soaking process, the crosslinking agent acts to combine elastin molecules with the surface of the metal substrate, a protein film is formed on the surface of the metal, and the elastin molecules self-assemble under the action of the HDI crosslinking agent to form a plurality of nucleation points inside the protein film, and the nucleation points can provide nucleation sites for the subsequent formation of fluorapatite.
After soaking, the Zr-based alloy with the protein film is immersed in the mineralized liquid for mineralization reaction, and a biological protein mineralized coating is formed on the surface of the Zr-based alloy. In the embodiment of the present invention, preferably, after the soaking is completed, the Zr-based alloy is taken out, washed with deionized water, and then the washed Zr-based alloy is immersed in the mineralized liquid. In the invention, the components of the mineralized liquid comprise sodium fluoride (NaF), hydroxyapatite (HAP) and water, the concentration of the sodium fluoride in the mineralized liquid is preferably 1-5 mmol/L, more preferably 1-3 mmol/L, and further preferably 2mmol/L, the concentration of the hydroxyapatite is preferably 1-5 mmol/L, more preferably 1-3 mol/L, and further preferably 2mmol/L, and the pH value of the mineralized liquid is preferably 5.5-6.5, and more preferably 6; in the specific embodiment of the invention, preferably, the sodium fluoride and the hydroxyapatite are added into deionized water, then nitric acid is added to accelerate the dissolution of the sodium fluoride and the hydroxyapatite, and after the sodium fluoride and the hydroxyapatite are completely dissolved, ammonia water is added to adjust the pH value of the solution to 5.5-6.5, so as to obtain the mineralized liquid; the nitric acid is preferably concentrated nitric acid (68 wt%), and the adding amount of the nitric acid is not particularly required and can be adjusted according to the amount of the mineralized liquid.
In the present invention, the temperature of the mineralization reaction is preferably 35 to 39 ℃, more preferably 37 ℃, the time of the mineralization reaction is preferably 6 to 10 days, more preferably 8 days, the mineralization reaction is preferably performed under a constant temperature condition, and in a specific embodiment of the present invention, the mineralization reaction is preferably performed in a constant temperature drying oven; in the specific embodiment of the invention, in the mineralization reaction process, the Zr-based alloy needs to be kept immersed in the mineralization liquid all the time, and when the liquid level of the mineralization liquid is lower than the surface of the Zr-based alloy, the mineralization reaction cannot be continued, and the reaction is stopped; the method provided by the invention can always ensure that the Zr-based alloy is submerged by the mineralized liquid in the mineralization reaction process, can ensure that the mineralization reaction is fully performed on the surface of the Zr-based alloy, and avoids the phenomenon of uneven mineralized coating. In the mineralization reaction process, sodium fluoride reacts with hydroxyapatite, and fluorapatite is formed from nucleation sites in the protein membrane, so that the biological protein mineralization coating is constructed.
After the mineralization reaction is finished, the bio-protein mineralization coating on the surface of the Zr-based alloy is preferably washed by using deionized water.
The invention also provides a Zr-based biomedical material, which comprises a Zr-based alloy and a bioprotein mineralization coating attached to the surface of the Zr-based alloy, wherein the bioprotein mineralization coating is constructed by the method in the scheme, the thickness of the bioprotein mineralization coating is preferably 1 mu m, the components of the Zr-based alloy are preferably consistent with the scheme, and the details are not repeated here. The invention constructs the biological protein mineralized coating on the surface of the Zr-based alloy, can greatly improve the biocompatibility of the Zr-based alloy, enables the Zr-based alloy to have certain bioactivity, and greatly improves the potential clinical use value of the Zr-based alloy in the aspect of medical materials.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the ELR protein used was purchased from Biotech, beijing Novoskrit, and the specific sequences are given above.
Example 1
In this embodiment, a Zr-based biomedical alloy is used as a substrate, and the Zr-based biomedical alloy comprises the following components in percentage by weight: nb:16% and the balance of zirconium and unavoidable impurity elements, and is recorded as Zr-16Nb alloy.
The method for constructing the mineralized coating on the surface of the Zr-16Nb alloy comprises the following steps:
(1) A Zr-16Nb alloy wafer was prepared for use as a base material.
(2) ELR protein is dissolved in a mixed solution of DMSO and DMF (the volume ratio of DMSO to DMF is 1:9), and the mixed solution is placed in a centrifuge to be stirred uniformly.
(3) Adding a cross-linking agent HDI into a mixed solution of ELR, DMF and DMSO, and uniformly mixing to obtain a protein solution, wherein the concentration of ELR protein in the protein solution is 10mg/mL, and the molar ratio of HDI to ELR protein is 30; immersing the Zr-16Nb alloy wafer by adopting a protein solution, standing for 12h, taking out after the immersion is finished, and washing by using deionized water.
(4) Dissolving NaF and HAP into deionized water, adding concentrated nitric acid and ammonia water, and adjusting the pH of the solution to 6 to obtain mineralized liquid, wherein the concentration of NaF in the mineralized liquid is 2mmol/L, and the concentration of HAP in the mineralized liquid is 2mmol/L; and (4) putting the Zr-16Nb alloy wafer washed in the step (3) into a mineralized liquid.
(5) And (4) putting the mineralized liquid and the Zr-16Nb alloy wafer in the step (4) into a constant-temperature drying box, and drying for 8 days at 37 ℃ to obtain the mineralized coating.
SEM examination of the surface of the unmineralized Zr-16Nb alloy (i.e., the substrate) and the mineralized Zr-16Nb alloy is shown in FIGS. 1-2. As can be seen from fig. 1-2, the surface of the unmineralized Zr-based alloy is smooth, and a gully-shaped coating is formed on the surface of the mineralized Zr-based alloy, so that the surface is more favorable for the absorption and differentiation of osteoblasts than a completely flat surface.
Scanning the mineralized Zr-16Nb alloy surface by using an X-ray diffractometer, comparing with a PDF phase card, and obtaining the result as shown in figure 3. As can be seen from fig. 3, the presence of fluorapatite phase in the coating indicates that a mineralized coating is indeed formed on the surface of the Zr-based alloy.
Example 2
In the embodiment, the Zr-based biomedical alloy is used as a substrate, and the Zr-based biomedical alloy comprises the following components in percentage by weight: nb:16%, ti:4 percent, and the balance of zirconium and inevitable impurity elements, and is marked as Zr-16Nb-4Ti alloy.
The method for constructing the mineralized coating on the surface of the Zr-16Nb-4Ti alloy comprises the following steps:
(1) A Zr-16Nb-4Ti alloy wafer was prepared for use as a base material.
(2) ELR protein is dissolved in a mixed solution of DMSO and DMF, and the mixed solution is placed in a centrifuge to be uniformly stirred.
(3) Adding a crosslinking agent HDI into a mixed solution of ELR, DMF and DMSO (the volume ratio of DMSO to DMF is 1:9), uniformly mixing to obtain a protein solution, wherein the concentration of ELR protein in the protein solution is 20mg/mL, and the molar ratio of HDI to ELR protein is 50; immersing the Zr-16Nb-4Ti alloy wafer by adopting a protein solution, standing for 12.5h, taking out after the immersion is finished, and washing by using deionized water.
(4) Dissolving NaF and HAP into deionized water, adding concentrated nitric acid and ammonia water, and adjusting the pH of the solution to 6 to obtain mineralized liquid, wherein the concentration of NaF in the mineralized liquid is 2mmol/L, and the concentration of HAP in the mineralized liquid is 2mmol/L; and (4) putting the Zr-16Nb-4Ti alloy wafer washed in the step (3) into a mineralization liquid.
(5) And (4) putting the mineralized liquid and the Zr-16Nb-4Ti alloy round pieces in the step (4) into a constant-temperature drying box, and drying for 8 days at 37 ℃ to obtain the mineralized coating.
Example 3
In the embodiment, the Zr-based biomedical alloy is used as a substrate, and the Zr-based biomedical alloy comprises the following components in percentage by weight: nb:16%, ti:8 percent, and the balance of zirconium and inevitable impurity elements, and is marked as Zr-16Nb-8Ti alloy.
The method for constructing the mineralized coating on the surface of the Zr-16Nb-8Ti alloy comprises the following steps:
(1) A Zr-16Nb-8Ti alloy wafer was prepared for use as a base material.
(2) ELR protein is dissolved in a mixed solution of DMSO and DMF (the volume ratio of the DMSO to the DMF is 1:9), and the mixed solution is placed in a centrifuge to be stirred uniformly.
(3) Adding a cross-linking agent HDI into a mixed solution of ELR, DMF and DMSO, and uniformly mixing to obtain a protein solution, wherein the concentration of ELR protein in the protein solution is 30mg/mL, and the molar ratio of HDI to ELR protein is 70; immersing the Zr-16Nb-8Ti alloy wafer by adopting a protein solution, standing for 13h, taking out after the immersion is finished, and washing by using deionized water.
(4) Dissolving NaF and HAP into deionized water, adding concentrated nitric acid and ammonia water, and adjusting the pH of the solution to 6 to obtain mineralized liquid, wherein the concentration of NaF in the mineralized liquid is 2mmol/L, and the concentration of HAP in the mineralized liquid is 2mmol/L; and (4) putting the Zr-16Nb-8Ti alloy wafer washed in the step (3) into a mineralization liquid.
(5) And (4) putting the mineralized liquid and the Zr-16Nb-8Ti alloy round pieces in the step (4) into a constant-temperature drying box, and drying for 8 days at 37 ℃ to obtain the mineralized coating.
Example 4
In the embodiment, the Zr-based biomedical alloy is used as a substrate, and the Zr-based biomedical alloy comprises the following components in percentage by weight: nb:16%, ti:12 percent, and the balance of zirconium and inevitable impurity elements, and is marked as Zr-16Nb-12Ti alloy.
The method for constructing the mineralized coating on the surface of the Zr-16Nb-12Ti alloy comprises the following steps:
(1) A Zr-16Nb-12Ti alloy wafer was prepared and used as a base material.
(2) ELR protein is dissolved in a mixed solution of DMSO and DMF (the volume ratio of DMSO to DMF is 1:9), and the mixed solution is placed in a centrifuge to be stirred uniformly.
(3) Adding a cross-linking agent HDI into a mixed solution of ELR, DMF and DMSO, and uniformly mixing to obtain a protein solution, wherein the concentration of ELR protein in the protein solution is 40mg/mL, and the molar ratio of HDI to ELR protein is 100; immersing the Zr-16Nb-12Ti alloy wafer by adopting a protein solution, standing for 13.5h, taking out after the immersion is finished, and washing by using deionized water.
(4) Dissolving NaF and HAP into deionized water, adding concentrated nitric acid and ammonia water, and adjusting the pH of the solution to 6 to obtain mineralized liquid, wherein the concentration of NaF in the mineralized liquid is 2mmol/L, and the concentration of HAP is 2mmol/L; and (4) putting the Zr-16Nb-12Ti alloy wafer washed in the step (3) into a mineralization liquid.
(5) And (4) putting the mineralized liquid and the Zr-16Nb-12Ti alloy round pieces in the step (4) into a constant-temperature drying box, and drying for 8 days at 37 ℃ to obtain the mineralized coating.
Example 5
In the embodiment, the Zr-based biomedical alloy is used as a substrate, and the Zr-based biomedical alloy comprises the following components in percentage by weight: nb:16%, ti:16 percent, and the balance of zirconium and inevitable impurity elements, and is marked as Zr-16Nb-16Ti alloy.
The method for constructing the mineralized coating on the surface of the Zr-16Nb-16Ti alloy comprises the following steps:
(1) A Zr-16Nb-16Ti alloy wafer was prepared for use as a base material.
(2) ELR protein is dissolved in a mixed solution of DMSO and DMF (the volume ratio of the DMSO to the DMF is 1:9), and the mixed solution is placed in a centrifuge to be stirred uniformly.
(3) Adding a cross-linking agent HDI into a mixed solution of ELR, DMF and DMSO, and uniformly mixing to obtain a protein solution, wherein the concentration of ELR protein in the protein solution is 50mg/mL, and the molar ratio of HDI to ELR protein is 120; immersing the Zr-16Nb-16Ti alloy wafer by adopting a protein solution, standing for 14h, taking out after the immersion is finished, and washing by using deionized water.
(4) Dissolving NaF and HAP into deionized water, adding concentrated nitric acid and ammonia water, and adjusting the pH of the solution to 6 to obtain mineralized liquid, wherein the concentration of NaF in the mineralized liquid is 2mmol/L, and the concentration of HAP in the mineralized liquid is 2mmol/L; and (4) putting the Zr-16Nb-16Ti alloy wafer washed in the step (3) into a mineralization liquid.
(5) And (4) putting the mineralized liquid and the Zr-16Nb-16Ti alloy round pieces in the step (4) into a constant-temperature drying box, and drying for 8 days at 37 ℃ to obtain the mineralized coating.
Example 6
Other conditions were the same as in example 1 except that the Zr-based alloy was replaced with Zr-8Nb-12Ti in the following proportions by weight: nb:8%, ti:12%, the balance being zirconium and unavoidable impurity elements.
Example 7
Other conditions were the same as in example 1 except that the Zr-based alloy was replaced with Zr-12Nb-8Ti in the following weight percentages: nb:12%, ti:8 percent, and the balance of zirconium and inevitable impurity elements.
SEM test and XRD detection are carried out on the mineralized coatings on the surfaces of the Zr-based alloys obtained in the examples 2-7, and the obtained results show that the mineralized coatings of fluorapatite phases are formed on the surfaces of the Zr-based alloys and the appearances of the mineralized coatings are similar to those of the mineralized coatings in the example 1.
Test example
The mineralized Zr-based alloy prepared in example 2 was subjected to cell experiments to evaluate the cellular compatibility of the mineralized Zr-based alloy, specifically, human osteosarcoma cells (MG 63), and the following steps were performed:
MG 63 osteosarcoma cells were cultured in MEM (minimum essential medium) medium supplemented with 10% Fetal Bovine Serum (FBS), 100. Mu.g/mL streptomycin and 100U/mL penicillin. Cell at 37 ℃ 5% CO 2 The culture medium is replaced every three days during the culture process. By observation, when the cells were grown to cover about 80% of the area of the culture dish, they were digested with 0.25% pancreatin, then the pancreatin was neutralized with serum, centrifuged, blown uniformly and prepared into a cell suspension, which was then passaged according to 1:5 and subjected to cell experiments using passage 3-5 cells. And (3) grinding and polishing two surfaces of Zr-based metal for experiments, then washing with alcohol, drying, and then carrying out ultraviolet lamp sterilization treatment, wherein each surface of the sample is irradiated for 12 hours.
After culturing 3-5 generation cells on the alloy surface for 24h, fixing the sample with 2.5% glutaraldehyde at 4 ℃ for 4h, then dehydrating with 20%, 40%, 60%, 80%, 100% gradient ethanol for 10min, then air-drying in the air for 1h, spraying gold on the sample, and observing cell adhesion by using SEM.
Fig. 4 is an SEM image of osteoblasts on the surface of the mineralized Zr-based alloy. According to the graph 4, the adhesion, proliferation and differentiation of osteoblasts on the surface of the mineralized coating are performed, and pseudopodia is grown, which shows that the compatibility of the Zr-based alloy material and cells can be obviously improved by constructing the mineralized coating.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. A method for constructing a biological protein mineralized coating on the surface of a Zr-based alloy is characterized by comprising the following steps:
(1) Soaking the Zr-based alloy in a protein solution to form a protein film on the surface of the Zr-based alloy; the protein solution comprises the components of elastin, a cross-linking agent and a solvent; the solvent in the protein solution is a mixed solvent of dimethyl sulfoxide and dimethylformamide, and the volume ratio of the dimethyl sulfoxide to the dimethylformamide in the mixed solvent is 1 (6 to 10); the cross-linking agent is hexamethylene diisocyanate, and the molar ratio of the cross-linking agent to the elastin is (30-150): 1; the Zr-based alloy comprises the following components in percentage by mass: 8 to 16 percent of Nb, 0 to 16 percent of Ti, and the balance of zirconium and inevitable impurities;
(2) Immersing the Zr-based alloy with the formed protein film into a mineralization liquid for mineralization reaction, and forming a biological protein mineralization coating on the surface of the Zr-based alloy; the components of the mineralized liquid comprise sodium fluoride, hydroxyapatite and deionized water.
2. The method of claim 1, wherein the sequence of elastin is MESLLP- [ ((VPGIG) 2VPGKG (VPGIG) 2) 2-DDDEEKFLRRIGRFG- ((VPGIG) 2VPGKG (VPGIG) 2) 2]3-V; the concentration of elastin in the protein solution is 1 to 100mg/mL.
3. The method according to claim 1 or 2, wherein the soaking in the step (1) is carried out at room temperature for 8 to 24h under a standing condition; the relative humidity of the environment during the soaking process is below 20%.
4. The method according to claim 1, wherein the concentration of sodium fluoride in the mineralized liquid is 1 to 5mmol/L, the concentration of hydroxyapatite is 1 to 5mmol/L, and the pH value of the mineralized liquid is 5.5 to 6.5.
5. The method according to claim 1, wherein the temperature of the mineralization reaction is 35 to 39 ℃ and the time is 6 to 10 days.
6. A Zr-based biomedical material comprising a Zr-based alloy and a bioprotein mineralization coating attached to the surface of the Zr-based alloy, said bioprotein mineralization coating being constructed by the method of any of claims 1~5.
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