CN114504678B

CN114504678B - Hydroxyapatite nanorod arrayed coating coated with graphene oxide and preparation method and application thereof

Info

Publication number: CN114504678B
Application number: CN202210152402.8A
Authority: CN
Inventors: 憨勇; 薛祯祯
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2023-02-17
Anticipated expiration: 2042-02-18
Also published as: CN114504678A

Abstract

The invention relates to a hydroxyapatite nanorod arrayed coating coated with graphene oxide, and a preparation method and application thereof ₃ COO) ₂ Ca、β‑C ₃ H ₇ Na ₂ O ₆ Micro-arc oxidation treatment is carried out in electrolyte of P and NaOH, and then the obtained amorphous TiO is treated ₂ The coating is immersed in a NaOH solution for hydrothermal reaction and then is treated with C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ 、β‑C ₃ H ₇ Na ₂ O ₆ Carrying out hydrothermal treatment on the mixed solution of P and NaOH; finally, the obtained nano-rod array coating of the hydroxyapatite is immersed in C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ 、β‑C ₃ H ₇ Na ₂ O ₆ Performing hydrothermal treatment in a mixed system of P, naOH and graphene oxide to obtain the hydroxyapatite nanorod arrayed coating coated with the graphene oxide.

Description

Hydroxyapatite nanorod arrayed coating coated with graphene oxide and preparation method and application thereof

Technical Field

The invention belongs to the field of surface micro-nano configuration and chemical component activation of biomedical materials, and particularly relates to a hydroxyapatite nanorod arrayed coating coated with graphene oxide, and a preparation method and application thereof.

Background

Hydroxyapatite (Ca) ₁₀ (PO ₄ ) ₆ (OH) ₂ HA) as the major inorganic component of human bone and teeth, HAs become the first choice biomaterial due to its similarity to human bone histochemical components and proximity of mineral phase configuration, and is commonly used in bone and tissue engineering in a variety of different forms and shapes. But its poor fracture toughness and brittleness limit its load bearing capacity after replacing bone tissue. To solve this problem, it was subsequently found that the HA-based composite material is not only excellent in bone conduction ability and bone-binding ability to adjacent tissues but also HAs no harmful effect on cells. Then Al ₂ O ₃ 、TiO ₂ 、ZrO ₂ CNT, etc. are gradually startedAre used as a reinforcement material for HA to improve its mechanical properties. However, reinforcing materials such as CNTs also often impair the biological activity and biological properties of HA or adversely affect adjacent tissues, and ideally reinforcing materials should impart mechanical integrity to the composite without reducing its biological activity. Graphene Oxide (GO) has attracted much attention due to the presence of functional groups such as epoxy, carbonyl, hydroxyl, and carboxyl groups, which are abundant on the surface. On one hand, the functional groups on the graphene oxide surface can provide a large number of reaction sites for chemical interaction, so that the graphene oxide sheet obtains high mechanical property, high hydrophilicity and good biocompatibility under the reduction states of high temperature, chemical treatment and the like; on the other hand, the presence of these functional groups makes the surface of the HA exhibit negative charges, which are compatible with Ca in HA ²⁺ Better adhesion. Therefore, the graphene oxide is an ideal and promising nano-scale reinforcing filler, and can improve the interface binding capacity and the biological activity of the composite material.

Moreover, bacterial infections occurring after annual joint replacement and fracture repair operations often cause implant failure, not only leading to revision of secondary operations, but also possible resulting in suppuration, even necessitating removal of the implant, excision of the infected tissue, with consequent economic burden and physical distress. Therefore, the ideal implant should promote bone tissue regeneration and prevent infection by drug-resistant bacteria. It is known that the inflammation reaction generated in the early stage of implanting biological material firstly occurs on the surface of the plant, so that the functionalized design of the surface of the plant can achieve the purpose of resisting infection. The process of bacterial killing is mainly related to physical or chemical damage to microorganisms. The edge of the graphene oxide nano sheet has the function similar to a blade/knife, and the outer bacterial membrane can be split. The outer membrane and cell wall of the bacterium are important components of the bacterium and are responsible for maintaining the normal shape and osmotic pressure of the bacterium and preventing the damage of mechanical stress, when the outer membrane and cell wall are subjected to the cleavage effect, the normal function of the outer membrane and cell wall can be obstructed, cytoplasmic components can be leaked, and GO further has the antibacterial and bactericidal effects. Second, GO acts as an oxidant to oxidize proteins and lipids in the bacterial molecule or to inhibit or kill bacteria by inactivating proteins via Reactive Oxygen Species (ROS) generated by light-dependent or light-dependent reactions, resulting in lipid peroxidation and destruction of nucleic acids. In addition, GO can permeate into the bacterial lipid membrane due to its high surface active sites and extract most of the phospholipids on the lipid membrane, so that the integrity of the lipid membrane and functions of the bacteria is destroyed and the bacteria die; besides the direct damage of GO to bacteria, GO can also have a wrapping effect on bacteria to isolate the bacteria from the normal growth and metabolism nutritional environment, so that the bacteria are inactivated. With the development of nanotechnology, the smaller the size area of the GO nano sheet is, the more the oxygen-containing active sites are, and the higher the oxidation antibacterial activity of GO independent of ROS is.

Thus, hydroxyapatite (HA) and Graphene Oxide (GO) nanocomposites are new biomaterials with biological properties that are more attractive than pure hydroxyapatite. Although a plurality of articles report the preparation method and biological effect of the HA-GO composite material, the biocompatibility and other performances of the hydroxyapatite-graphene oxide nanocomposite material still need to be improved, and the report of the nanocomposite material in the aspect of immunity and antibiosis is not seen.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a hydroxyapatite nanorod arrayed coating coated with graphene oxide and a preparation method and application thereof, wherein an HA arrayed structure and GO active molecules on the surface of the coating are utilized to promote adhesion, proliferation and differentiation on cells and promote collagen secretion of the cells, so that the generation of new bones around an implant material is remarkably promoted, and a good integration effect between the implant material and bone tissues is realized; meanwhile, better antibacterial action of the implant material is realized by means of the oxidation action of the graphene oxide on substances in bacteria, the extraction action on RNA in a bacterial lipid membrane and the cutting action of the edge of the graphene oxide on the bacteria, so that the problem of implant failure caused by related infection and insufficient osteogenic activity of the implant is solved.

The invention is realized by the following technical scheme:

a preparation method of a hydroxyapatite nanorod arrayed coating coated with graphene oxide comprises the following steps:

step 1, titanium or titanium alloy is added into a mixture Containing (CH) ₃ COO) ₂ Ca、β-C ₃ H ₇ Na ₂ O ₆ Micro-arc oxidation treatment is carried out in electrolyte of P and NaOH to obtain amorphous TiO containing calcium and phosphorus ₂ Coating;

step 2, firstly, amorphous TiO containing calcium and phosphorus ₂ Immersing the coating in NaOH solution with the concentration of 0.01-0.025M for hydrothermal reaction, and then coating C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ 、β-C ₃ H ₇ Na ₂ O ₆ Carrying out hydrothermal treatment at 100-140 ℃ in a mixed solution of P and NaOH to obtain a hydroxyapatite-containing nanorod arrayed coating;

step 3, immersing the hydroxyapatite nanorod array coating in the step 2 in the solution C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ 、β-C ₃ H ₇ Na ₂ O ₆ In a mixed system of P, naOH and graphene oxide, the graphene oxide and beta-C in the mixed solution in the step 2 ₃ H ₇ Na ₂ O ₆ The proportion of P is (0.2-1.8) g: (0.3-0.45) mmol, and carrying out hydrothermal treatment at 110-140 ℃ to obtain the hydroxyapatite nanorod arrayed coating coated with the graphene oxide.

Preferably, in the electrolyte solution described in step 1, (CH) ₃ COO) ₂ The concentration of Ca is 0.2-0.3M, beta-C ₃ H ₇ Na ₂ O ₆ The concentration of P is 0.02-0.03M, and the concentration of NaOH is 0.005-0.01M.

Preferably, the titanium or titanium alloy in step 1 is rod-shaped, cuboid or disk.

Preferably, the micro-arc oxidation treatment in the step 1 is performed by using a direct current pulse power supply, wherein the positive voltage is 350-450V, the negative voltage is 50-100V, the frequency is 80-120 Hz, and the duty ratio is 10% -20%.

Further, the micro-arc oxidation treatment adopts titanium or titanium alloy as an anode, stainless steel as a cathode, the distance between the cathode and the anode is 10-15 cm, and the micro-arc oxidation treatment is carried out for 5-10 min under the conditions.

In a preferred embodiment of the method of the invention,TiO described in step 2 ₂ The coating is subjected to hydrothermal treatment in NaOH solution at 80-120 ℃ for 2-6 h.

Preferably, in the mixed solution of step 2, C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ The concentration of (A) is 0.1-0.15M, beta-C ₃ H ₇ Na ₂ O ₆ The concentration of P is 0.02-0.03M, the concentration of NaOH is 0.125-0.175M, and the coating obtained in the step 2 is hydrothermally treated for 6-12 h at 100-140 ℃ in the mixed solution.

Preferably, in the mixed system described in step 3, C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ In a concentration of 0.02M, beta-C ₃ H ₇ Na ₂ O ₆ The concentration of P is 0.04M, the concentration of NaOH is 0.025M, and the nano-rod array coating of hydroxyapatite is hydrothermally treated for 6 to 12 hours at 110 to 140 ℃ in a mixed system.

Preferably, the graphene oxide in step 3 is obtained by the following process:

according to the weight ratio of (0.4-0.8) g: (100-200 mL), firstly oxidizing and stripping conductive graphite powder and concentrated nitric acid at 110-150 ℃ for 24-48 h, then purifying graphene oxide in the reaction solution to obtain a clarified solution, carrying out vacuum drying on the clarified solution, and grinding and refining the obtained solid to obtain graphene oxide powder.

The hydroxyapatite nanorod arrayed coating with the graphene oxide coated on the titanium base surface, which is obtained by the preparation method of the hydroxyapatite nanorod arrayed coating with the graphene oxide coated, is provided.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention discloses a preparation method of a hydroxyapatite nanorod arrayed coating coated with graphene oxide, which HAs the properties of vascularization, osteogenic differentiation and antibiosis of Graphene Oxide (GO), wherein a functional coating capable of forming bones and antibiosis is constructed on the surface of titanium or alloy in a mode of combining micro-arc oxidation and hydrothermal treatment, the functional coating exists in a mode that GO is uniformly wrapped on the surface of HA, GO wrapped on the surface layer is distributed in a yarn-shaped thin layer, and HA on the subsurface layer is in a hexagonal prism-shaped structureUniformly distributed in the amorphous TiO ₂ TiO on the inner layer of the hard ceramic film ₂ The titanium alloy is continuously distributed on the surface of titanium or alloy in a porous form, and the bioactivity of the surface of the titanium alloy is improved through the dual actions of surface configuration and chemical components. The method has the advantages of simple and controllable process, high repeatability, convenient operation, economy and environmental protection, controllable components of the micro-arc oxidation electrolyte and the hydrothermal solution, difficult decomposition, capability of realizing the control of the components and the performance of the film layer by adjusting the component proportion of the solution, high film forming speed and film forming thickness of the titanium dioxide oxide layer prepared by the micro-arc oxidation method, metallurgical bonding with a substrate, high bonding strength and difficult peeling in the process of being implanted into a body; the coating generated on the titanium surface HAs high hardness and good corrosion resistance comprehensive performance indexes, the uniform coating of the yarn-shaped graphene oxide thin layer on HA is successfully realized, and the surface appearance of HA is not changed. The coating prepared by the method is firmly combined with the matrix, and the problem of peeling of the implant material caused by micromotion can be well avoided when the implant material is stressed and moves. The HA-GO nano composite material is prepared by combining double factors of physicochemical property and configuration, so that an important support is provided for the feasibility of the GO-based nano composite material in immune antibiosis.

The hydroxyapatite nanorod arrayed coating coated with the graphene oxide, which is prepared by the invention, is internally provided with compact ceramic phase TiO ₂ The oxidation layer is formed, the corrosivity of the titanium or titanium alloy substrate in body fluid can be well improved, the hexagonal prism-shaped HA on the subsurface layer can promote new osteogenesis through a configuration effect, and the GO wrapped on the surface layer can better play roles of angiogenesis and antibiosis while promoting the new osteogenesis.

Drawings

FIG. 1 is a TEM image of graphene oxide synthesized by an oil bath method in the present invention;

FIG. 2 is an infrared spectrum of graphene oxide after 48h oxidation of graphite in an oil bath according to the present invention;

FIG. 3 shows the micro-arc oxidized porous TiO of the present invention ₂ SEM image of surface topography of layer (example 4 coating);

FIG. 4 is an SEM image of the surface morphology of the HA nanorod arrayed coating of the invention after micro-arc oxidation and hydrothermal treatment of titanium and its alloy at 1 μm (example 4 coating);

FIG. 5 is an SEM image of the surface morphology of the HA nanorod arrayed coating of the invention after micro-arc oxidation and hydrothermal treatment of titanium and its alloy at 20 μm (example 4 coating);

FIG. 6a is a micro-topography of HG1 under SEM corresponding to 10 μm;

FIG. 6b is a microscopic morphology of HG1 under SEM corresponding to 500 nm;

FIG. 6c is a microscopic morphology of HG1 under SEM corresponding to 200 nm;

FIG. 6d is a micro-topography of HG5 under SEM corresponding to 10 μm;

FIG. 6e is a microscopic morphology of HG5 under SEM at 500 nm;

FIG. 6f is a microscopic morphology of HG5 under SEM at 200 nm;

FIG. 6g is a microscopic morphology of HG9 under SEM corresponding to 10 μm;

FIG. 6h is a microscopic morphology of HG9 at 500nm under SEM;

FIG. 6i is a microscopic morphology of HG9 at 200nm under SEM,

FIG. 7 is an infrared spectrum of the composite coating after HA is uniformly coated with graphene oxide according to the present invention;

FIG. 8a is a TEM image of the microstructure of the composite coating after HA is uniformly coated with graphene oxide in the invention;

FIG. 8b is a high resolution image of the composite coating after HA is uniformly coated with graphene oxide according to the present invention;

FIG. 8c is an electron diffraction spot diagram of the composite coating after HA is uniformly coated with graphene oxide according to the present invention;

FIG. 9 is a diagram of the cell viability of rat bone marrow stromal stem cells (BMSCs) in accordance with the present invention after co-culture with different target composite coatings;

FIG. 10 is a graph of collagen secretion in rat bone marrow stromal stem cells (BMSCs) co-cultured with different target composite coatings in accordance with the present invention.

Detailed Description

The invention will be further elucidated with reference to the embodiments described hereinafter, which are to be regarded as illustrative rather than restrictive, and with reference to the accompanying drawings.

The invention relates to a preparation method of a hydroxyapatite nanorod arrayed coating coated with graphene oxide, which comprises the following steps:

(1) Oil Bath (OB) preparation of graphene oxide

Measuring 0.4-0.8 g of conductive graphite powder and 100-200 mL of concentrated nitric acid, transferring the conductive graphite powder and the concentrated nitric acid into a conical flask containing a rotor, putting the conical flask into an oil bath pan, adjusting the stirring speed of the solution in the oil bath pan at 20-25 r/min, oxidizing and stripping the solution for 24-48 h at 110-150 ℃, opening a condensing tube during reaction, cooling and collecting volatilized gas-phase concentrated nitric acid, and dissipating part of heat in the reaction process;

after the reaction is finished, centrifuging the reaction solution, taking upper-layer clarified liquid, then repeatedly centrifuging the upper-layer clarified liquid to purify GO in the clarified liquid, transferring the purified clarified liquid into a mortar for vacuum drying, and grinding and refining the dried sample to obtain GO nano powder with a lamellar structure;

(2) Micro-arc oxidation (MAO) of titanium or titanium alloy

Preparing (CH) with a concentration of 0.2-0.3M ₃ COO) ₂ Ca solution, beta-C of 0.02-0.03M ₃ H ₇ Na ₂ O ₆ P (beta-GP) solution and 0.005-0.01M NaOH solution are taken as electrolyte, titanium or titanium alloy and stainless steel are respectively taken as anode and cathode in the micro-arc oxidation device and are immersed in the electrolyte, the titanium or titanium alloy is rod-shaped, cuboid or wafer, and the micro-arc oxidation treatment is carried out on the titanium or titanium alloy through the action of electrochemical reaction and plasma discharge;

the specific process parameters of the micro-arc oxidation are as follows: adopting a direct current pulse power supply, controlling the positive voltage to be 350-450V, controlling the negative voltage to be 50-100V, setting the frequency to be 80-120 Hz, setting the duty ratio to be 10-20%, controlling the distance between a cathode plate and an anode plate to be 10-15 cm, and then carrying out micro-arc oxidation treatment on titanium or titanium alloy for 5-10 minutes, wherein Ca in the electrolyte can be treated by violent plasma discharge reaction in the micro-arc oxidation process ²⁺ 、PO ₄ ^3- Embedding into titanium oxide coating to obtain amorphous TiO containing Ca and P elements ₂ Coating;

(3) Hydrothermal Treatment (HT) to obtain hydroxyapatite nanorod array

With TiO containing Ca, P elements ₂ The coating is taken as a substrate and is immersed into a 15mL hydrothermal reaction lining of NaOH solution with the concentration of 0.01-0.025M to react for 2-6 h at the temperature of 80-120 ℃, and in the hydrothermal reaction process, amorphous TiO containing Ca and P elements ₂ Ca in the coating ²⁺ 、PO ₄ ^3- Will be continuously precipitated on the surface of the coating, and can be mixed with OH in the hydrothermal solution after the surface of the coating is gathered to reach the critical concentration ^- Combined and Ca with the prolongation of hydrothermal reaction time ²⁺ 、PO ₄ ^3- Continuously precipitate to obtain amorphous TiO ₂ Obtaining HA nucleation points on the surface of the coating; then putting the mixture into 15mL of C with the concentration of 0.1-0.15M ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ (Ca-EDTA), 0.02-0.03M beta-GP and 0.125-0.175M NaOH, performing hydrothermal treatment at 100-140 ℃ for 6-12 h, and then performing Ca, P and OH treatment ^- Continuously supplying, and obtaining the HA nanorod array coating which is uniformly distributed, complete in configuration and hexagonal prism-shaped.

(4) Preparation of hydroxyapatite nanorod arrayed coating coated with graphene oxide

Placing the HA nanorod arrayed coating obtained above in the inner liner of a reaction kettle, and adding 15mL of C with the concentration of 0.02M ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ (Ca-EDTA), 0.04M beta-GP, 0.025M NaOH mixed solution and 0.2-1.8 g of GO powder, so that the HA nanorod arrayed coating is immersed in the solution and reacts for 6-12 h at 110-150 ℃ to obtain a target composite coating (according to the dosage change of GO in a sample, the samples are sequentially named HG1, HG5 and HG 9);

the surface layer of the composite coating obtained by the method is a uniformly distributed graphene oxide yarn-shaped thin layer, and the thickness of a single-layer GO thin layer is only about 0.34nm; the subsurface layer is HA nanorod array, the nanorods are hexagonal prism-shaped, and the diameter of the nanorods is 120-140nm, 400-600 nm in length, and the inner layer (near the substrate layer) is dense amorphous porous TiO in continuous distribution ₂ The base layer is titanium or titanium alloy. The coating is endowed with high osseointegration and antibacterial performance through the synergistic effect of materials among layers, which enables the bone tissue engineering to play an important role in clinical application.

Example 1

(1) Oil Bath (OB) preparation of graphene oxide

0.4g of conductive graphite powder is measured, 100mL of concentrated nitric acid is put into a conical flask, then the conical flask is put into an oil bath pot, oxidized and stripped for 48 hours at the temperature of 110 ℃, a condensing tube is started to cool and collect volatilized gas-phase concentrated nitric acid while the reaction is heated, and partial reaction heat is dissipated.

And after the reaction is finished, moving the liquid in the conical flask into a centrifugal tube, separating and taking the supernatant part in the product five times at the rotating speed of 10000r/min, wherein the supernatant part in the centrifugal tube is clear and bright reddish brown, transferring the centrifugal tube into a mortar for vacuum drying treatment for 10 hours, and grinding and refining the dried sample to obtain the GO nano powder with the lamellar structure.

The infrared spectrogram of graphene oxide is shown in FIG. 2, in which 1708cm ^-1 、1632cm ^-1 、1452cm ^-1 Stretching vibration peak of C = O in GO corresponding to peak at position, sp ² Stretching vibration peak hybridized with C = C and deformation vibration peak of OH in C-OH, 1348cm ^-1 The peak at the position is the vibration peak of C-O in carboxyl, 1384cm ^-1 、1242cm ^-1 、1051cm ^-1 The positions of the C-O-H deformation vibration peak, the C-OH expansion vibration peak and the C-O expansion vibration peak are respectively corresponding to 1147cm ^-1 The position corresponds to the characteristic peak of C-O-C in GO, and 862cm ^-1 And then the peak is the vibration peak of epoxy group in GO, and in conclusion, after graphite is oxidized, a plurality of functional groups such as-COOH, -OH, -C = O, -CH (O) CH-and the like appear, namely the synthesized target GO powder. Meanwhile, the appearance of the film under a transmission electron microscope corresponds to that of the film shown in the figure 1, the surface of the film is flat and smooth, the film is a transparent yarn-shaped thin layer, the thickness of the film is only 1.038nm, and the folded part in the figure is formed by rolling and shrinking a stable self-structure for reducing the surface energy.

(2) Micro-arc oxidation (MAO) of titanium or titanium alloy

Preparing a solution Containing (CH) with a concentration of 0.2M ₃ COO) ₂ Ca solution, 0.02M beta-C ₃ H ₇ Na ₂ O ₆ P (beta-GP) solution and 0.005M NaOH solution are compounded and used as electrolyte, pure metal titanium sheets and stainless steel are respectively used as an anode and a cathode in a micro-arc oxidation device and are placed in the electrolyte, a direct current pulse power supply is adopted, plasma discharge under high temperature and high pressure is utilized, micro-arc oxidation treatment is carried out on titanium for 5min under the conditions that positive voltage is 450V, negative voltage is 50V, frequency is 100Hz, duty ratio is 10 percent, and distance between a cathode plate and an anode plate is 10cm, so as to obtain amorphous TiO containing Ca and P elements ₂ The surface appearance of the hard ceramic coating is in a porous structure and is distributed on the surface of the titanium substrate, referring to figure 3, and the surface of the amorphous oxide film layer is provided with a uniform micron-sized blind hole structure.

(3) Nucleation and initial growth of Hydrothermal Treatment (HT) hydroxyapatite nanorod array

Adding TiO containing Ca and P elements ₂ The coating is placed in a 50mL reaction kettle, the filling degree is 30%, the hydrothermal solution with the concentration of 0.01M NaOH reacts for 6 hours at the temperature of 80 ℃, and then nucleation sites of HA which are uniformly distributed are obtained on the amorphous coating. Subsequently, the mixture was adjusted to a concentration of 15mL to 0.1M C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ In a hydrothermal mixed salt solution of (Ca-EDTA), 0.02M beta-GP and 0.125M NaOH, the degree of filling of the reaction kettle is 30%, and after hydrothermal treatment at 100 ℃ for 12h, the coating with the structure shown in FIG. 4 is obtained, which is a high-power morphology diagram of the HA nanorod array coating with a hydrothermal growing configuration of a hexagonal prism structure under a Scanning Electron Microscope (SEM), and FIG. 5 is a corresponding low-power morphology diagram.

The HA nanorod arrayed coating is placed in the inner liner of a 50mL reaction kettle, and 0.02M C is added into the reaction kettle ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ (Ca-EDTA), 0.04M beta-GP, a hydrothermal mixed solution of 0.025M NaOH and 1g of GO powder with a filling degree of 30%, and reacting at 110 ℃ for 12h to obtain the HG5 target composite coatingAnd (3) a layer.

Fig. 6 is a microscopic morphology of a hydroxyapatite nanorod arrayed coating coated with different graphene oxides, in which HA is hexagonal prism-shaped and approximately perpendicular to the substrate surface, GO is a yarn-shaped thin layer coated on the HA surface, and the thickness of a single layer of GO is only 0.34nm, which is more beneficial to the interaction with cells at the later stage and promotes the adhesion, proliferation and differentiation of stem cells. From fig. 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h and 6i, it can be seen that after GO is compounded with HA, the diameter is 120-140 nm, the length is 400-600 nm, and among them, the coating of HA by graphene oxide in HG5 coating is most uniform.

The infrared spectrogram of the target composite coating respectively corresponds to the curves HG1, HG5 and HG9 of FIG. 7, analysis shows that the infrared spectrograms of different coatings have no obvious difference, and the infrared spectrograms are illustrated by taking HG5 as an example, and can be known from the graph at 1708cm ^-1 、1452cm ^-1 、1348cm ^-1 The peak at the position respectively corresponds to a C = O stretching vibration peak in GO, a deformation vibration peak in C-OH and a C-O vibration peak in COOH, which indicates the existence of GO in the composite coating; at the same time, 2922cm ^-1 And 2852cm ^-1 The peak at the position corresponds to-CH inherent in the reduced graphene oxide ₂ Characteristic absorption peaks of GO, indicating some reduction of GO during hydrothermal process, which indirectly verifies presence of GO in the composite. And 1052cm ^-1 、572cm ^-1 The peaks at the positions respectively correspond to v 3PO ₄ ^3- Asymmetric stretching vibration peak sum v 4PO ₄ ^3- The peak of stretching vibration of (a), which indicates the presence of HA in the composite coating; in conclusion, HG composite coatings have been successfully prepared by the present invention.

Fig. 8a is a tissue morphology diagram of the HG5 composite coating under TEM (transmission electron microscope), and fig. 8b is a high resolution image of the composite coating, wherein the composite coating contains an HA phase and a GO species, as seen from the distance d =0.345nm between adjacent crystal planes and the thickness d =0.34nm between adjacent amorphous layers; meanwhile, by combining each crystal face of HA corresponding to the electron diffraction spots of HA in FIG. 8c, it can be shown that the coating is an HG composite coating.

Example 2

(1) Oil Bath (OB) preparation of graphene oxide

0.4g of conductive graphite powder is measured, 100mL of concentrated nitric acid is put into a conical flask, then the conical flask is put into an oil bath pot, oxidized and stripped for 36 hours at 130 ℃, a condensing tube is started to cool and collect volatilized gas-phase concentrated nitric acid while the reaction is heated, and part of reaction heat is dissipated.

And after the reaction is finished, moving the liquid in the conical flask into a centrifugal tube, separating and taking the supernatant part in the product for four times at the rotating speed of 10000r/min, transferring the clear liquid in the centrifugal tube into a mortar for vacuum drying treatment for 10 hours, and grinding and refining the dried sample to obtain the GO nano powder with a lamellar structure.

(2) Micro-arc oxidation (MAO) of titanium alloy

Preparing a solution Containing (CH) with a concentration of 0.25M ₃ COO) ₂ Ca solution, 0.025M beta-C ₃ H ₇ Na ₂ O ₆ Taking a compound solution of a P (beta-GP) solution and a 0.0075M NaOH solution as an electrolyte, taking a rod-shaped pure metal titanium sheet and stainless steel as an anode and a cathode respectively in a micro-arc oxidation device, placing the anode and the cathode in the electrolyte, adopting a direct current pulse power supply, performing micro-arc oxidation treatment on titanium for 5min by utilizing plasma discharge under high temperature and high pressure under the conditions of positive voltage of 400V, negative voltage of 75V, frequency of 80Hz, duty ratio of 15 percent and distance between a cathode plate and an anode plate of 10cm to obtain amorphous TiO containing Ca and P elements ₂ Hard ceramic coating.

(3) Nucleation and initial growth of Hydrothermal (HT) hydroxyapatite nanorod array

Adding TiO containing Ca and P elements ₂ The coating is placed in a 50mL reaction kettle, the filling degree is 30%, the hydrothermal solution with the concentration of 0.015M NaOH reacts for 4 hours at the temperature of 100 ℃, and then nucleation sites of HA which are uniformly distributed are obtained on the amorphous coating. Then put it into 15mL of 0.125M C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ In a hydrothermal mixed salt solution of (Ca-EDTA), 0.025M beta-GP and 0.15M NaOH, the filling degree of the reaction kettle is 30%, and after hydrothermal treatment at 120 ℃ for 9 hours, the hydrothermal growing HA nanorod array coating is obtained, wherein the structure of the coating is a hexagonal prism nanorod.

Placing the HA nanorod arrayed coating in the inner liner of a 50mL reaction kettle, and adding C with the concentration of 0.02M ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ (Ca-EDTA), 0.04M beta-GP, 0.025M NaOH and 0.2g of GO powder with a filling degree of 30%, and reacting for 9h at 130 ℃ to obtain the HG1 target composite coating.

Example 3

(1) Oil Bath (OB) preparation of graphene oxide

0.4g of conductive graphite powder is measured, 100mL of concentrated nitric acid is put into a conical flask, then the conical flask is put into an oil bath pan, the conical flask is oxidized and stripped for 24 hours at the temperature of 150 ℃, a condensing tube is opened to cool and collect volatilized gas-phase concentrated nitric acid while the conical flask is heated in a reaction, and part of reaction heat is dissipated.

And after the reaction is finished, moving the liquid in the conical flask into a centrifugal tube, separating and taking the supernatant part in the product for six times at the rotating speed of 10000r/min, transferring the supernatant part in the centrifugal tube into a mortar for vacuum drying treatment for 10 hours, and grinding and refining the dried sample to obtain the GO nano powder with a lamellar structure.

(2) Micro-arc oxidation (MAO) of titanium alloy

Preparing a mixture Containing (CH) with a concentration of 0.3M ₃ COO) ₂ Ca solution, 0.03M beta-C ₃ H ₇ Na ₂ O ₆ A compound solution of a P (beta-GP) solution and a 0.01M NaOH solution is used as an electrolyte, a cuboid titanium alloy and stainless steel are respectively used as an anode and a cathode in a micro-arc oxidation device and are placed in the electrolyte, a direct current pulse power supply is adopted, plasma discharge under high temperature and high pressure is utilized, and micro-arc oxidation treatment is carried out on titanium for 5min under the conditions that the positive voltage is 350V, the negative voltage is 100V, the frequency is 120Hz, the duty ratio is 20 percent, and the distance between a cathode plate and an anode plate is 10cm, so as to obtain amorphous TiO containing Ca and P elements ₂ Hard ceramic coating.

Will contain Ca and P elementsOf TiO (2) ₂ The coating is placed in a 50mL reaction kettle, the filling degree is 30%, the hydrothermal solution with the concentration of 0.02M NaOH reacts for 2 hours at 120 ℃, and then nucleation sites of HA which are uniformly distributed are obtained on the amorphous coating. Followed by bringing it to a concentration of 15mL to 0.15M C ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ In a hydrothermal mixed salt solution of (Ca-EDTA), 0.03M beta-GP and 0.175M NaOH, the filling degree of the reaction kettle is 30%, and after hydrothermal treatment at the temperature of 140 ℃ for 6 hours, a hydrothermal long HA nanorod array coating with a hexagonal prism-shaped nanorod configuration is obtained.

Placing the HA nanorod arrayed coating in the inner liner of a 50mL reaction kettle, and adding C with the concentration of 0.02M ₁₀ h ₁₂ CaN ₂ Na ₂ O ₈ (Ca-EDTA), 0.04M beta-GP, a hydrothermal mixed solution of 0.025M NaOH and 1.8g of GO powder with a filling degree of 30%, and reacting at 150 ℃ for 6h to obtain the target HG9 composite coating.

The cell experiments were performed on the target composite coating as follows.

Experiment 1

Will contain 2X 10 ⁴ The cell suspension/mL is inoculated into a 24-well plate containing a sample in sequence, after complete culture for 1, 3 and 7 days, the activity and proliferation of rat bone marrow stromal stem cells (BMSCs) after coculture with target composite coatings HG1, HG5 and HG9 are measured by an Alamar Blue staining method, and the results show that: compared with the HA control group, the experimental group HG composite coating prepared by the method HAs a remarkable promoting effect on the cell activity of the BMSCs so as to indirectly show the promoting effect of the target composite coating on the growth and proliferation of the BMSCs, wherein the promoting effect is most remarkable in the experimental group HG5, and the reference is shown in FIG. 9;

experiment 2

Will contain 2X 10 ⁴ The cell suspension of/mL is inoculated into a 24-well plate containing samples in sequence, after complete culture for 3, 7 and 14 days, the cells are stained by sirius red, then the dye on the surface of the stained sample is eluted, and the absorbance of the sample is measured at 540nm to explore the rBMSCs glue on the surfaces of different coatingsThe effect of the original secretion is to promote the generation of mineralized bone at the later stage, and fig. 10 is a result graph of the effect, since sirius red is a strong acid dye, it can react with collagen fibers to cause the collagen fibers to generate a significant birefringence phenomenon, and thus the collagen fibers are shown to be red. Therefore, by analyzing the color of the dyed collagen fibers, the adsorption of the surfaces of the HG experimental group materials prepared by the method of the invention to the dye is stronger compared with that of the HA control group, wherein the accelerating effect of the HG5 group is most obvious;

the embodiments may be listed in many, not limited to space, and not individually listed here.

The above contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention should not be limited thereby, and any modification made on the basis of the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a hydroxyapatite nanorod arrayed coating coated with graphene oxide is characterized by comprising the following steps:

step 1, titanium or titanium alloy is added into a mixture Containing (CH) ₃ COO) ₂ Ca、β-C ₃ H ₇ Na ₂ O ₆ Performing micro-arc oxidation treatment in electrolyte of P and NaOH, wherein the titanium or titanium alloy is rod-shaped, cuboid or wafer in the electrolyte (CH) ₃ COO) ₂ The concentration of Ca is 0.2-0.3M, beta-C ₃ H ₇ Na ₂ O ₆ The concentration of P is 0.02-0.03M, the concentration of NaOH is 0.005-0.01M, and amorphous TiO containing calcium and phosphorus is obtained ₂ Coating;

the micro-arc oxidation treatment is carried out by adopting a direct current pulse power supply, the positive voltage is 350-450V, the negative voltage is 50-100V, the frequency is 80-120 Hz, the duty ratio is 10-20%, titanium or titanium alloy is adopted as an anode, stainless steel is adopted as a cathode, the distance between the cathode and the anode is 10-15 cm, and the micro-arc oxidation treatment is carried out for 5-10 min under the conditions;

step 2, firstly, amorphous TiO containing calcium and phosphorus ₂ The coating is immersed in NaOH solution with the concentration of 0.01 to 0.025M for hydrothermal reaction for 2 to 6 hours at the temperature of between 80 and 120 ℃,the resulting coating is then coated at C ₁₀ H ₁₂ CaN ₂ Na ₂ O ₈ 、β-C ₃ H ₇ Na ₂ O ₆ Hydrothermal reaction for 6-12h at 100-140 deg.c in the mixed liquid of P and NaOH ₁₀ H ₁₂ CaN ₂ Na ₂ O ₈ Has a concentration of 0.1-0.15M, beta-C ₃ H ₇ Na ₂ O ₆ The concentration of P is 0.02-0.03M, the concentration of NaOH is 0.125-0.175M, and a nano-rod array coating containing hydroxyapatite is obtained;

step 3, immersing the hydroxyapatite nanorod array coating in the step 2 in the solution C ₁₀ H ₁₂ CaN ₂ Na ₂ O ₈ 、β-C ₃ H ₇ Na ₂ O ₆ In a mixed system of P, naOH and graphene oxide, C ₁₀ H ₁₂ CaN ₂ Na ₂ O ₈ In a concentration of 0.02M, beta-C ₃ H ₇ Na ₂ O ₆ The concentration of P is 0.04M, the concentration of NaOH is 0.025M, and the graphene oxide and the beta-C in the mixed solution in the step 2 ₃ H ₇ Na ₂ O ₆ The proportion of P is (0.2-1.8) g: (0.3-0.45) mmol, and carrying out hydrothermal treatment at 110-140 ℃ for 6-12 h to obtain a hydroxyapatite nanorod arrayed coating coated with graphene oxide;

the graphene oxide is obtained by the following steps:

2. A hydroxyapatite nanorod arrayed coating coated with graphene oxide, obtained by the preparation method of the hydroxyapatite nanorod arrayed coating coated with graphene oxide according to claim 1.

3. The use of the graphene oxide coated hydroxyapatite nanorod arrayed coating of claim 2 in an implant material.