阅读说明：本技术 一种具有良好生物抗氧化功能的金属离子掺杂二氧化锰涂层及其制备方法和应用 (Metal ion doped manganese dioxide coating with good biological oxidation resistance function and preparation method and application thereof ) 是由 李恺 刘诗伟 谢有桃 邵丹丹 郑学斌 于 2019-11-04 设计创作，主要内容包括：本发明涉及一种具有良好生物抗氧化功能的金属离子掺杂二氧化锰涂层及其制备方法和应用,所述具有良好生物抗氧化功能的金属离子掺杂二氧化锰涂层包括：医用基材、以及形成在所述医用基材表面的具有纳米片或纳米颗粒状结构的金属离子掺杂二氧化锰涂层,所述金属离子掺杂二氧化锰涂层中金属离子为过渡金属离子；所述金属离子掺杂二氧化锰涂层中金属离子的掺杂量为5～40wt%。(The invention relates to a metal ion doped manganese dioxide coating with good biological oxidation resistance, a preparation method and application thereof, wherein the metal ion doped manganese dioxide coating with good biological oxidation resistance comprises the following components: the composite material comprises a medical base material and a metal ion doped manganese dioxide coating with a nano-sheet or nano-granular structure, wherein the metal ion doped manganese dioxide coating is formed on the surface of the medical base material, and the metal ion in the metal ion doped manganese dioxide coating is a transition metal ion; the doping amount of the metal ions in the metal ion doped manganese dioxide coating is 5-40 wt%.)

1. A metal ion doped manganese dioxide coating with good biological oxidation resistance is characterized by comprising: the composite material comprises a medical base material and a metal ion doped manganese dioxide coating with a nano-sheet or nano-granular structure, wherein the metal ion doped manganese dioxide coating is formed on the surface of the medical base material, and the metal ion in the metal ion doped manganese dioxide coating is a transition metal ion; the doping amount of the metal ions in the metal ion doped manganese dioxide coating is 5-40 wt%.

2. The metal ion doped manganese dioxide coating of claim 1, wherein the metal ion is Fe^{3 +}、Co²⁺、Ni²⁺、Cu²⁺And Zn²⁺At least one of (1).

3. The metal ion doped manganese dioxide coating of claim 1 or 2, wherein the manganese dioxide in the metal ion doped manganese dioxide coating is birnessite type manganese dioxide.

4. The metal ion doped manganese dioxide coating according to any of claims 1-3, wherein the medical substrate is selected from medical metal substrates, or medical alloy substrates, preferably pure titanium, titanium alloys, stainless steel, or cobalt chromium molybdenum alloys.

5. A method for preparing metal ion doped manganese dioxide coating with good biological oxidation resistance according to any one of claims 1 to 4, which comprises:

(1) dissolving potassium permanganate serving as a manganese source and a metal ion salt serving as a doping source in deionized water to obtain a mixed solution;

(2) growing a metal ion doped manganese dioxide coating on the surface of the medical base material in situ by adopting a solution reaction method; the solution reaction method is an ultraviolet light reduction method.

6. The preparation method according to claim 5, wherein the concentration of potassium permanganate in the mixed solution is 0.001-0.1 mol/L, and the concentration of metal ions is 0.5-30% of potassium permanganate.

7. The method according to claim 5 or 6, wherein the parameters of the UV photo-reduction method include: the power of the ultraviolet lamp is 20-60W; the distance between the lamp source and the liquid level of the mixed solution is 2-10 cm; the ultraviolet irradiation time is 8-24 hours.

8. Use of a metal ion doped manganese dioxide coating with good biological oxidation resistance as defined in any one of claims 1 to 4 in the preparation of hard tissue repair and replacement biomaterials.

Technical Field

The invention relates to a biological antioxidant coating under oxidative stress environment, a preparation method and application thereof, in particular to a metal ion doped manganese dioxide biological antioxidant coating with good biological antioxidant function, a preparation method and application thereof, and belongs to the technical field of biomedicine.

Background

With the acceleration of the aging process of the population and the increase of bone injury accidents caused by traffic accidents, diseases, natural disasters and the like, the demand of artificial bone implant materials is increasing day by day. Compared with the bone defect patients in normal physiological states, in the microenvironment in vivo of the patients with systemic diseases (such as diabetes, hypertension, osteoporosis and the like), the active oxygen clusters (mainly comprising hydrogen peroxide molecules, superoxide radical ions and hydroxyl free radicals) have higher level, so that the oxidation capacity of the organism exceeds the oxidation resistance, the oxidative stress damage of tissues around the bone implant material is easily caused, the activity of osteoblasts is inhibited, and the repair of the bone tissues after the operation is seriously influenced. Therefore, the development of bone implant materials with good biological oxidation resistance has important clinical significance for promoting bone repair under oxidative stress and improving bone quality.

The metallic titanium material and the alloy thereof are common bone implant materials and have good biocompatibility, chemical stability and corrosion resistance. However, the titanium-based medical materials commonly used in clinic do not have biological oxidation resistance, and cannot meet the requirement of effective repair of bone tissues under oxidative stress. Currently, cerium oxide (CeO) is prepared by plasma spray techniques₂) The biological coating has certain anti-oxidation stress performance (J Mater Sci: Mater Med (2016)27: 100). But the preparation cost of the coating is high, the process parameters are complex, the coating does not have a nano structure, and the capability of removing active oxygen clusters such as hydrogen peroxide is limited. Manganese dioxide (MnO)₂) Is a transition metal oxide material with low price and easy preparation. The manganese dioxide nanoenzyme has better hydrogen peroxide scavenging capacity and shows the over-mentioned propertyMimic activity of Catalase. Mn in the presence of ligands such as phosphate or carbonate²⁺Has certain capacity of eliminating superoxide radical ion. And Mn²⁺Is an important cofactor of superoxide dismutase (MnSOD), and can resist oxidative stress injury of cells. In bone metabolism, Mn²⁺Can participate in the synthesis of glycoprotein required by cartilage and bone formation, and has important function for maintaining normal skeletal development. In addition, it has been found that MnO can be increased by doping metal ions₂The catalytic performance of the composite material is further improved, the catalytic activity of the composite material is expected to be improved, and the composite material is expected to have better oxidation resistance, so that the possibility is provided for researching and developing novel efficient and cheap biological oxidation resistant coatings.

Disclosure of Invention

In order to solve the problems of the defects in the prior art, the invention aims to provide a coating with good biological oxidation resistance, and a preparation method and application thereof.

In one aspect, the present invention provides a metal ion doped manganese dioxide coating with good biological oxidation resistance, comprising: the composite material comprises a medical base material and a metal ion doped manganese dioxide coating with a nano-sheet or nano-granular structure, wherein the metal ion is formed on the surface of the medical base material and is a transition metal ion; the doping amount of the metal ions in the metal ion doped manganese dioxide coating is 5-40 wt%.

In the invention, the manganese dioxide coating doped with metal ions (transition metal ions) is grown in situ on the surface of the base material of the medical metal or medical alloy material to serve as a biological coating, so that the biological oxidation resistance of the medical base material is improved. Compared with the undoped manganese dioxide coating, the metal ion doped manganese dioxide coating obtained by the invention has better hydrogen peroxide scavenging capacity.

Preferably, the metal ion is Fe³⁺、Co²⁺、Ni²⁺、Cu²⁺And Zn²⁺At least one of (1).

Preferably, the manganese dioxide in the metal ion doped manganese dioxide coating is birnessite type manganese dioxide.

Preferably, the medical substrate is selected from a medical metal substrate or a medical alloy substrate, and is preferably pure titanium, a titanium alloy, stainless steel or a cobalt-chromium-molybdenum alloy.

On the other hand, the invention provides a preparation method of a metal ion doped manganese dioxide coating with good biological oxidation resistance, which comprises the following steps:

(1) dissolving potassium permanganate serving as a manganese source and a metal ion salt serving as a doping source in deionized water to obtain a mixed solution;

(2) growing a metal ion doped manganese dioxide coating on the surface of the medical base material in situ by adopting a solution reaction method; the solution reaction method is an ultraviolet light reduction method.

Preferably, the concentration of the potassium permanganate in the mixed solution is 0.001-0.1 mol/L, and the concentration of the metal ions is 0.5-30% of that of the potassium permanganate.

Preferably, the parameters of the ultraviolet light reduction method include: the power of the ultraviolet lamp is 20-60W; the distance between the lamp source and the liquid level of the mixed solution is 2-10 cm; the ultraviolet irradiation time is 8-24 hours. The ultraviolet photoelectrons generated in the ultraviolet irradiation reduction method can reduce the activation energy required by the potassium permanganate reaction and promote the potassium permanganate and the matrix material to generate the manganese dioxide coating through the oxidation reduction reaction.

On the other hand, the invention provides the application of the metal ion doped manganese dioxide coating with good biological oxidation resistance in preparation of hard tissue repair and replacement biomaterials. The coating obtained by the invention has good biocompatibility and biological oxidation resistance, can effectively reduce oxidative stress damage of bone cells, promotes the repair of bone tissues under an oxidative stress environment, is a potential biomedical material, and can be used for hard tissue repair and the research and development of replacing biological materials.

Has the advantages that:

the invention provides MnO doped with metal ions₂The coating has good biocompatibility and biological oxidation resistance, can effectively reduce oxidative stress damage of bone cells, and promotes the repair of bone tissues in an oxidative stress environment; also, the preparation method of the inventionThe method has the advantages of low cost, simple operation, good repeatability, suitability for large-scale production and the like.

Drawings

FIG. 1 shows MnO collected from the reaction solution after the coating preparation is finished₂And Fe³⁺Doping MnO₂(Fe-MnO₂)、Co²⁺Doping MnO₂(Co-MnO₂)、Ni²⁺Doping MnO₂(Ni-MnO₂)、Cu²⁺Doping MnO₂(Cu-MnO₂) And Zn²⁺Doping MnO₂(Zn-MnO₂) XRD spectrum of the powder;

FIG. 2 shows MnO₂Coating and Fe-MnO₂、Co-MnO₂、Ni-MnO₂、Cu-MnO₂And Zn-MnO₂XPS full spectrum of the coating;

FIG. 3 shows MnO₂Coating and Fe-MnO₂、Co-MnO₂、Ni-MnO₂、Cu-MnO₂And Zn-MnO₂SEM photograph of the coating;

FIG. 4 shows MnO₂Coating and Fe-MnO₂、Co-MnO₂、Ni-MnO₂、Cu-MnO₂And Zn-MnO₂The coating decomposes hydrogen peroxide to generate oxygen;

FIG. 5 shows cells in MnO under normal culture conditions₂Coating and Zn-MnO₂Cell proliferation (a), alkaline phosphatase (B) and extracellular matrix mineralization (C) of the coated surface;

FIG. 6 shows cells in MnO under hydrogen peroxide simulated oxidative stress environment₂Coating and Zn-MnO₂Cell proliferation (a), alkaline phosphatase (B) and extracellular matrix mineralization (C) of the coated surface.

Detailed Description

The present invention is further illustrated by the following examples, which are to be understood as merely illustrative and not restrictive.

In the present disclosure, the provided biological oxidation resistant coating refers to metal ion doped MnO grown in situ on the surface of the medical substrate₂A coating having a nanosheet structure or a nanoparticulate shape. The metal ion doping with good biological oxidation resistanceThe metal ion in the manganese dioxide coating may be Fe³⁺、Co²⁺、Ni²⁺、Cu²⁺、Zn²⁺The transition metal ions have good biocompatibility and biological oxidation resistance, and can effectively promote the bone tissue repair in oxidative stress environment. MnO₂The crystal form of (a) may be a birnessite type. The coating can be formed on common medical metal or medical alloy materials such as pure titanium, titanium alloy, stainless steel or cobalt-chromium-molybdenum alloy. Such a matrix material may provide better processability and mechanical properties.

In an alternative embodiment, the doping amount of the metal ions in the metal ion doped manganese dioxide coating is 5-40 wt%.

In one embodiment of the invention, the MnO doped with metal ions is grown in situ on the surface of the base material by a solution reaction method which is simple to operate and can be produced in large scale₂Coating to obtain the bone implantation coating material with good biological oxidation resistance. The method has the advantages of low cost, simple operation, good repeatability, suitability for large-scale production and the like.

The following is an exemplary description of a method for preparing a metal ion doped manganese dioxide coating layer having a good biological oxidation resistance.

In the present invention, MnO doped with metal ion₂The coating is prepared by adopting an ultraviolet illumination reduction method.

Ultraviolet light reduction method. Potassium permanganate is used as a manganese source, metal ion salts are used as doping sources, and the potassium permanganate and the metal ion salts are dissolved in deionized water to obtain a mixed solution. Then the medical base material is put into the mixed solution and reduced for a certain time under ultraviolet light to obtain MnO doped with metal ions₂And (4) coating. The parameters of the ultraviolet light reduction method comprise: the power of the ultraviolet lamp is 20-60W; the distance between the lamp source and the liquid level of the mixed solution is 2-10 cm; the ultraviolet irradiation time is 8-24 hours. Wherein the concentration of the potassium permanganate in the mixed solution can be 0.001-0.1 mol/L, and the doping concentration of the metal ions is 0.5-30% of the concentration of the potassium permanganate.

In the present invention, the metal ion salt employed may include Fe³⁺、Co²⁺、Ni²⁺、Cu²⁺、Zn²⁺Nitrate, sulfate, chloride, etc. of transition metal ions.

The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.

Example 1

A: preparation of metal ion doped MnO by ultraviolet light reduction method₂Coating layer

Placing titanium sheet polished smooth by abrasive paper in potassium permanganate and metal ions for doping (the raw materials for doping metal ions are ferric trichloride (FeCl)₃) Cobalt nitrate (Co (NO)₃)₂) Nickel chloride (NiCl)₂) Copper sulfate (CuSO)₄) And zinc chloride (ZnCl)₂) In situ growth of metal ion doped nano MnO on the surface of the titanium sheet by irradiating the mixed solution under ultraviolet light for a period of time₂And (4) coating.

Wherein the ion mixing volume is 20mL, the concentration of the used potassium permanganate is 0.01M, and the concentration of the metal ion solution for doping is 0.002M. The power of an ultraviolet lamp is 48W, the distance between a lamp source and the liquid level of the mixed solution is about 5cm, the irradiation time of the ultraviolet light is 12h, and MnO is respectively obtained₂Coating, Fe-MnO₂Coating (Fe content 33.1 wt%), Co-MnO₂Coating (Co content 37.4 wt%), Ni-MnO₂Coating (Ni content 20.5 wt%), Cu-MnO₂Coating (Cu content 25.1 wt.%) and Zn-MnO₂Coating (Zn content 16.8 wt%). The doping amount of the metal ions is obtained according to the ICP-OES test result.

And after the preparation of the coating is finished, performing phase analysis on the powder sample collected in the reaction solution. As shown in FIG. 1, MnO₂And Fe-MnO₂，Co-MnO₂，Ni-MnO₂，Cu-MnO₂And Zn-MnO₂The samples are all birnessite type MnO₂(JCPDS 80-1098), no other impurity peaks were detected. Wherein Fe-MnO₂And Co-MnO₂The degree of crystallinity of the sample was better than that of undoped MnO₂Sample, and Ni-MnO₂，Cu-MnO₂And Zn-MnO₂The sample has a lower degree of crystallinity than undoped MnO₂And (3) sampling.

As can be seen from the XPS survey shown in fig. 2, the sample mainly contained: the adsorbed C element, the Ti element in the base material, the Mn element, the O element and various doped elements in the coating show that the elements of Fe, Co, Ni, Cu, Zn and the like are successfully doped into corresponding ion-doped MnO₂In the sample.

As can be seen from the SEM scan shown in FIG. 3, MnO was present₂And Fe-MnO₂，Co-MnO₂，Ni-MnO₂，Cu-MnO₂And Zn-MnO₂The coating is of a nano-sheet or nano-particle structure.

B: MnO doped with metal ions₂Detection of performance of catalytic decomposition of hydrogen peroxide by coating

The sample was immersed in a 0.15mM hydrogen peroxide solution using a volume of 4mL, and the concentration of oxygen in the solution was monitored in real time using a dissolved oxygen meter. The higher the relative concentration of oxygen in the solution, the greater the ability of the sample to catalytically decompose hydrogen peroxide. From the relative oxygen content in the solution as shown in FIG. 4, MnO was added to the titanium substrate₂MnO doped with metal ion₂The sample can obviously catalyze and decompose the hydrogen peroxide. And the MnO can be obviously improved by doping metal ions₂Catalytic properties of the coating, thus MnO doped with metal ions₂The coating has better hydrogen peroxide removing capability.

C：Zn-MnO₂Biocompatibility of the coating and facilitating bone Performance testing

Mouse preosteoblasts MC3T3-E1 were used for cell proliferation, differentiation and mineralization experiments:

(1) cell proliferation

The samples were sterilized using a steam sterilizer (121 ℃, 30min), and each set of sterile materials was carefully placed in a 48-well cell culture plate. Collected and grownThe MC3T3-E1 cells in good state were digested and the cell suspension concentration was adjusted. 1mL of cell suspension (10000 cells/mL) was seeded on the sample surface, and a final concentration of 0.15mM hydrogen peroxide was added to a portion of the wells. At 37 deg.C, 5% CO₂After culturing in the cell culture box for 1, 4 and 7 days, respectively, the culture solution was discarded. 1mL of fresh medium and 0.1mL of CCK-8 solution were added to each well. 37 ℃ and 5% CO₂After further incubation for 3h in the cell incubator, the well solutions were carefully aspirated and added to the 96-well plates. Measuring the OD value of each hole at 450nm by using an enzyme-labeling instrument;

(2) cell differentiation: quantitative determination of alkaline phosphatase

The samples were sterilized using a steam sterilizer (121 ℃, 30min), and each set of sterile materials was carefully placed in a 48-well cell culture plate. The MC3T3-E1 cells with good growth state are collected, digested and the concentration of the cell suspension is adjusted. 1mL of cell suspension (50000 cells/mL) was seeded onto the sample surface and a final concentration of 0.15mM hydrogen peroxide was added to a portion of the wells. At 37 deg.C, 5% CO₂After culturing for 7 and 14 days in the cell culture box, the culture medium was discarded. mu.L of 0.1% Triton X-100(PBS diluted) was added to each well. After the surface of the sample is blown by repeatedly sucking with a gun head, the liquid in the hole and the foam are sucked into an EP tube together for centrifugation (10000 revolutions, 5 min). Chromogenic substrate solutions and standard working solutions (0.5mM) were prepared, and blank control wells, standard wells, and sample wells were set using 96-well plates with reference to Table 1. The amounts of standards were 4, 8, 16, 24, 32 and 40 μ L, respectively. After mixing the liquid with the aid of a shaker (50rpm/min), incubation was carried out for 10min at 37 ℃. mu.L of stop solution was added to each well, and absorbance was measured at 405nm using a microplate reader. After normalization of total protein concentration, ALP quantitation was obtained:

table 1 is an illustration of the placement of the blank control wells, standard wells and sample wells:

	blank control	Standard article	Sample (I)
				Detection buffer solution	50μL	100-x	-
Chromogenic substrates	50μL	-	50
				Sample (I)	-	-	50
Working solution for standard substance	-	x	-

；

(3) Cell mineralization: alizarin red staining quantitative detection

The samples were sterilized using a steam sterilizer (121 ℃, 30min), and each set of sterile materials was carefully placed in a 48-well cell culture plate. The MC3T3-E1 cells with good growth state are collected, digested and the concentration of the cell suspension is adjusted. 1mL of cell suspension (50000 cells/mL) was seeded onto the sample surface and a final concentration of 0.15mM hydrogen peroxide was added to a portion of the wells. At 37 deg.C, 5% CO₂After 14 and 21 days of culture in the cell incubator, respectively, the culture medium was discarded. Each well was filled with 1mL of 4% polymethyl methacrylateAldehyde solution, 4 degrees C were incubated for 15 min. The supernatant was discarded, washed with PBS 3 times, and 500. mu.L of alizarin red dye solution was added to each well, followed by incubation at room temperature for 20 min. After discarding the supernatant, washing 3 times with PBS, 500. mu.L of 10% cetylpyridinium chloride solution was added to each well and incubated at room temperature for 15 min. OD was measured at 590nm using a microplate reader.

As can be seen from FIG. 5, under normal culture conditions, MnO was present₂Coating and Zn-MnO₂The coating has good biocompatibility, and the preosteoblasts of MC3T3-E1 are in MnO₂And Zn-MnO₂The proliferation, differentiation and mineralization abilities of the surface of the coating are obviously better than those of a substrate control group. And Zn-MnO₂The coating promotes proliferation, differentiation and mineralization of pre-MC 3T3-E1 osteoblasts due to MnO₂And (4) coating.

As can be seen from FIG. 6, under the hydrogen peroxide-simulated oxidative stress condition, MC3T3-E1 preosteoblasts were in MnO₂And Zn-MnO₂The proliferation, differentiation and mineralization capability of the surface of the coating is obviously superior to that of a substrate control group, which shows that MnO is₂And Zn-MnO₂The coating has good biological oxidation resistance and bone promoting ability, wherein Zn-MnO is₂The biological oxidation resistance and bone-promoting capability of the coating are superior to MnO₂And (4) coating.