CN113018510B

CN113018510B - Method for modifying surface of titanium-based implant and composite coating on surface of titanium-based implant

Info

Publication number: CN113018510B
Application number: CN202110141639.1A
Authority: CN
Inventors: 刘宣勇; 马小涵; 谭继
Original assignee: Shanghai Institute of Ceramics of CAS
Current assignee: Shanghai Institute of Ceramics of CAS
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2022-08-09
Anticipated expiration: 2041-02-02
Also published as: CN113018510A

Abstract

The invention relates to a method for modifying the surface of a titanium-based implant and a composite coating on the surface of the titanium-based implant, wherein the composite coating on the surface of the titanium-based implant comprises an inner layer, a middle layer and an outer layer which are sequentially formed on a substrate of the titanium-based implant; the inner layer is formed inwards from the surface of the titanium-based implant along the thickness direction, and M metal material exists in the forms of simple substance of M and oxide of M, wherein M is calcium and/or strontium; an M metal material exists in the intermediate layer in the form of a simple substance of M and an oxide of M, and an N metal material exists in the form of an oxide of N, wherein N is at least one of magnesium, calcium, zinc, sodium and potassium; the outer layer comprises an oxide of N; and controlling the degradation time of the N oxide in the outer layer under the physiological environment by regulating the thickness of the outer layer film.

Description

Method for modifying surface of titanium-based implant and composite coating on surface of titanium-based implant

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a method for carrying out surface modification on a titanium-based orthopedic implant and a composite coating on the surface of the titanium-based implant.

Background

Due to the increasing number of patients with dental defects, fractures, osteoporosis and bone tumors, there is a great market demand for hard tissue implants. Titanium and its alloy are the first choice materials for current hard tissue substitutes and restorations by virtue of their excellent biocompatibility, corrosion resistance and good mechanical properties.

However, the failure of the implantation operation still occurs, and long-term clinical studies find that the amount of the key problems restricting the success rate and the service life of the clinical operation of the titanium-based implant is as follows (progress Materials Science,2009,54: 397-: on one hand, the surface of the titanium-based implant has no antibacterial performance, and bacteria are easy to adhere and reproduce on the surface to form a biological film at the initial stage of implantation, so that the bacterial infection related to the implant frequently occurs; on the other hand, titanium is a biological inert material, which causes unsatisfactory bone performance, fails to form effective osseointegration between bone and implant, and is prone to implant loosening in the later stage of implantation. Therefore, the growth of bacteria on the surface of the titanium-based implant can be inhibited in the early stage, and the growth of the osteoblast-related cells is promoted in the later stage.

At present, the modification strategies for improving the antibacterial performance of the surface of the titanium-based implant mainly comprise two strategies: firstly, antibiotics are loaded on the surface; secondly, loading inorganic antibacterial agent. However, abuse of antibiotics can lead to the development of bacterial resistance; antibacterial agents (such as Ag, Zn, Cu, etc.) also have significant toxic side effects on normal cells and tissues. Therefore, there is a need to develop safer and more effective antibacterial strategies.

Disclosure of Invention

Aiming at the problems that the prior titanium-based implant clinically used is easy to generate bacterial infection in the initial stage of implantation and implant loosening in the later stage of implantation due to insufficient antibacterial and osteogenic properties, the invention aims to provide a titanium surface (titanium-based implant surface) modification method so as to meet the requirements of better antibacterial property in the initial stage and better bone property in the later stage of medical titanium implant materials.

The present inventors have conducted extensive and intensive studies in order to achieve the above object. The energy metabolism of bacteria is mainly dependent on the normal operation of the respiratory electron transport chain on the bacterial membrane. Bacteria produce transmembrane H by respiration ⁺ Concentration gradients, thus forming transmembrane proton motive potentials, driving the synthesis of "energy currency" ATP (adenosine triphosphate) (Annual Review of Biochemistry 1997,66, 717-749.). Recent studies have shown that the local alkaline microenvironment of the material surface can consume extracellular H of bacteria ⁺ Inhibiting the growth of bacteria; the more basic the surface microenvironment, the greater the corresponding antibacterial properties (ACS applied Materials)&Interfaces 2018,10, 42018-. In contrast, the respiratory electron transport chain of mammalian cells is locatedIntracellular mitochondrial membranes are less affected by the alkaline microenvironment than bacteria. And the activity of alkaline phosphatase (ALP) can be enhanced by a proper alkalescent microenvironment, and the proliferation of osteoblasts and the differentiation of mesenchymal stem cells of bone marrow can be promoted (Journal of Materials Chemistry,2012,22,8662), so that the new bone generation is facilitated. Metals such as Mg/Ca and their metal oxides can react with water to form OH ^- Creating an alkaline microenvironment; the Mg/Ca ions generated at the same time can promote the adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells (Acta biomaterials, 2014,10,2834-2842, Bone,2010,46, 571-576).

Based on the consideration, a coating or a film capable of forming an alkaline microenvironment is constructed on the surface of the medical titanium, so that the osteogenesis promoting effect is expected to be generated, the energy metabolism of bacteria can be inhibited, the antibacterial effect is generated, and the stronger the alkalinity is, the better the antibacterial effect is. However, the excessively alkaline surface microenvironment, while killing bacteria, is also significantly toxic to cells (Journal of Colloid and Interface Science 2014,436, 160-170). For example, in physiological environments, magnesium alloy surfaces react with water to create an excessively strong alkaline microenvironment, which results in the inability of osteoblasts to adhere and grow well on the surface (ACS applied Materials & Interfaces 2016,8, 35033-. The present inventors have diligently studied that the above problems can be solved by the invention described below, thereby completing the present invention.

In a first aspect, the invention provides a titanium-based implant surface composite coating, which comprises an inner layer, a middle layer and an outer layer which are sequentially formed on a titanium-based implant substrate;

the inner layer is formed inwards from the surface of the titanium-based implant along the thickness direction, and M metal material exists in the forms of a simple substance of M and an oxide of M, wherein M is calcium and/or strontium;

an M metal material exists in the intermediate layer in the form of a simple substance of M and an oxide of M, and an N metal material exists in the form of an oxide of N, wherein N is at least one of magnesium, calcium, zinc, sodium and potassium;

the outer layer comprises an oxide of N;

and controlling the degradation time of the N oxide in the outer layer under the physiological environment by regulating the thickness of the outer layer film.

The invention provides a modification strategy for improving the antibacterial and osteogenic properties of a coating (film) capable of forming an alkaline microenvironment on the surface of a titanium-based implant. Under physiological environment, the composite coating can generate a strong alkaline surface microenvironment containing M (also called as 'first metal') and N (also called as 'second metal') ions (such as magnesium and calcium ions) at the initial stage, has good antibacterial performance, and can promote mineralization of bone marrow mesenchymal stem extracellular matrix; with the gradual degradation of the second metal oxide (such as magnesium oxide), a weak alkaline surface microenvironment containing the first metal ions (such as calcium ions) can be generated at the later stage, which has good bone-promoting performance and can inhibit the growth of bacteria. The degradation time can be regulated and controlled within a certain thickness range through the second metal oxide (such as magnesium oxide), the surface antibacterial effect of the titanium-based implant is ensured, and various clinical requirements are met.

The thickness of the outer layer is 1-1000 nm.

The M atomic percent of the inner layer can be less than 20%, and the thickness of the inner layer can be 1-150 nm.

The thickness of the intermediate layer can be 1-120 nm.

In the intermediate layer, the distribution of titanium element increases progressively in the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, the distribution of oxygen element increases progressively first and then decreases progressively in the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, the distribution of M element increases progressively in the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, and the distribution of N element decreases progressively in the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer.

The titanium-based implant can be a titanium metal material, a titanium metal material with a titanium oxide coating on the surface, or a titanium metal material with at least one of a micron-scale structure, a submicron-scale structure and a nanometer-scale structure on the surface.

The titanium metal material may be pure titanium or a titanium alloy.

In a second aspect, the present invention provides a method for modifying the surface of a titanium-based implant, comprising forming the above-described titanium-based implant surface composite coating by:

(1) implanting M ions into the surface of the titanium-based implant substrate by adopting a plasma immersion ion implantation technology to construct an inner layer;

(2) and depositing N oxide on the surface of the inner layer by adopting a magnetron sputtering technology to construct and obtain the middle layer and the outer layer.

The step (1) may include: using M simple substance or M-Ti alloy as cathode, setting the temperature of a vacuum chamber for plasma immersion ion injection to be 10-100 ℃, and the background vacuum degree to be 1 x 10 ^-3 ～6×10 ^-3 Pa, the injection voltage is-5 to-50 kV, the frequency is 5 to 20Hz, the pulse width is 100 to 1000 mus, and the injection time is 0.5 to 240 minutes.

The step (2) may include: applying radio frequency magnetron sputtering on the target material, setting the temperature of a vacuum chamber for magnetron sputtering to be 10-100 ℃, and the background vacuum degree to be 1 multiplied by 10 ^-3 ～6×10 ^-3 Pa, radio frequency power of 100-600W, working pressure of 1 × 10 ^-1 20Pa and 30-240 minutes of sputtering time.

The titanium-based implant substrate may be formed by at least one of grinding, turning, etching, additive manufacturing techniques such as 3D printing, contouring, stereolithography, prior to step (1) (prior to disposing the composite coating). The surface of the titanium-based implant substrate may also be pretreated prior to implantation, the pretreatment including polishing and cleaning, or polishing and cleaning followed by grit blasting.

The medical titanium surface composite coating has the advantage that the alkalinity of the surface microenvironment of the medical titanium surface composite coating is changed in a time sequence manner under the physiological environment, and the staged antibacterial osteogenesis effect can be generated. In the initial stage, the second metal oxide (such as MgO) on the outer layer of the composite coating can rapidly react with water to form a strong alkaline microenvironment, so that a good antibacterial effect is generated, and bacterial infection in the initial stage of implantation is avoided. Further, consumption of the outer second metal oxide exposes the intermediate layer, either partially or completely, to the physiological environment, and the intermediate layer begins to participate in the reaction of the physiological environment. The intermediate layer can release the first metal ions (such as Ca ions), improve the biocompatibility of the basic microenvironment generated by the second metal oxide, and promote the adhesion, proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells. After the middle layer is consumed, the M-Ti-O (first metal-Ti-O) layer in the later-stage composite coating can slowly react with water for a long time to form a weak alkaline microenvironment containing first metal ions, so that the proliferation of bacteria is inhibited, the adhesion, the proliferation and the osteogenic differentiation of bone marrow mesenchymal stem cells are promoted, and finally good osseointegration is realized.

The modified titanium material obtained by modification treatment of the invention can obtain good osteogenic effect, and simultaneously can obtain good antibacterial effect by constructing an alkaline coating by using biologically safe first metal (such as Ca) and second metal (such as Mg) elements, thereby avoiding adverse effects of common antibacterial components on cell osteogenic activity. And the thickness of a second metal oxide (such as MgO) film and the doping depth and content of the first metal can be controlled by adjusting the technological parameters such as magnetron sputtering and ion implantation, so that the alkalinity and the duration of the surface microenvironment can be regulated and controlled, various actual clinical requirements can be met, and the problems of insufficient antibacterial performance and osseointegration capability of the titanium implant in clinic at present can be solved.

Drawings

FIG. 1 is a scanning electron microscope image of the surface of pure titanium and titanium-based material obtained by modification treatment in example 1;

FIG. 2 is the XRD spectrum of the titanium-based material and pure titanium surface element obtained by modification treatment in example 1;

in FIG. 3, a is the XPS spectrum of the modified titanium-based material and pure titanium obtained in example 1, and b is the high resolution spectrum of Ca on the surface of the Ca-Ti sample; c is the high resolution spectrum of Mg on the surface of the MgO-Ti sample; d is a high-resolution spectrum of Mg on the surface of the MgO @ Ca-Ti sample;

FIG. 4 is a graph of the depth distribution of elements in the titanium-based material obtained by the modification treatment of example 1

FIG. 5 shows the results of Ca and Mg ion release of the titanium-based material obtained by the modification treatment of example 1;

FIG. 6 is a graph showing the results of pH values of the entire solution after soaking the titanium-based material obtained by the modification treatment of example 1 and pure titanium in physiological saline for 24 hours;

FIG. 7 is a scanning electron micrograph of Staphylococcus aureus after 24 hours of surface culture of the titanium-based material and pure titanium obtained by modification treatment in example 1 and a fluorescence photograph of viable and dead stain (bacteria inoculation concentration of 10) ⁷ cfu/ml)；

FIG. 8 is a colony agar plate of Staphylococcus aureus (bacteria inoculation concentration of 10) after 24 hours of surface culture of the titanium-based material and pure titanium obtained by the modification treatment of example 1 ⁷ cfu/ml)；

FIG. 9 is a colony agar plate of Staphylococcus aureus (bacteria inoculation concentration of 10) after 24 hours of surface culture of the titanium-based material and pure titanium obtained by the modification treatment of example 1 ⁶ cfu/ml)；

FIG. 10 shows the antibacterial effects of the titanium-based material obtained by the modification treatment of example 1 and pure titanium against Staphylococcus aureus after immersion treatment in physiological saline for 1,4 and 7 days (bacterial inoculation concentration of 10) ⁷ cfu/ml), the right graph corresponds to the quantitative analysis result of the antibacterial rate of the titanium-based material obtained by the modification treatment of the embodiment 1;

fig. 11 is a photograph of cytoskeleton staining of bone marrow mesenchymal stem cells on the surface of titanium-based material and pure titanium obtained by modification treatment in example 1 for 1,4 and 24 hours.

FIG. 12 is a scanning electron microscope picture of mesenchymal stem cells after culturing for 7 days on the surface of the titanium-based material and pure titanium obtained by modification treatment in example 1;

FIG. 13 shows osteoblast gene expression results of mesenchymal stem cells after culturing on the surface of the titanium-based material and pure titanium obtained by modification treatment in example 1 for 7 days and 14 days;

FIG. 14 is a scanning electron microscope picture of mesenchymal stem cells and Staphylococcus aureus after co-culturing for 48 hours on the surface of the titanium-based material and pure titanium obtained by the modification treatment of example 1;

FIG. 15 is a photograph of live and dead staining of mesenchymal stem cells of bone marrow and Staphylococcus aureus after co-culturing for 48 hours on the surface of the titanium-based material and pure titanium obtained by the modification treatment of example 1;

FIG. 16 is a photograph showing a cross-section of a magnesium oxide film on the surface of samples of MgO-Ti-1, MgO-Ti-2 and MgO-Ti-3 according to an embodiment;

FIG. 17 shows the Mg ion release results for the MgO-Ti-1, MgO-Ti-2, and MgO-Ti-3 samples described in FIG. 16.

Detailed Description

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

The present disclosure relates to a method of surface modification of a titanium-based implant based on a titanium metal material and a modified titanium-based implant (hereinafter, sometimes simply referred to as "modified titanium") obtained. The titanium-based implant surface composite coating is formed by combining an inner layer which is formed on a titanium-based implant substrate and inwards forms from the titanium-based implant surface along the thickness direction and is provided with a first metal material in the form of a first metal simple substance and a first metal oxide, an intermediate layer which is provided with the first metal material in the form of the first metal simple substance and the first metal oxide and is provided with a second metal material in the form of a second metal oxide, and an outer layer which comprises the second metal oxide, so that the titanium-based implant surface composite coating is endowed with staged antibacterial and bone-promoting properties. Under physiological environment, the composite coating can generate a strong alkaline surface microenvironment containing a first metal (such as calcium ions) and a second metal ion (such as magnesium ions) at the initial stage, has good antibacterial performance, and can promote the mineralization of the bone marrow mesenchymal stem extracellular matrix; along with the gradual degradation of the second metal oxide, a weak alkaline surface microenvironment containing the first metal ions can be generated in the later period, so that the bone property is well promoted, and the growth of bacteria can be inhibited. Further, the inner layer is formed inward in the thickness direction from the surface of the titanium-based implant, and the surface of the inner layer can also be regarded as the surface of the titanium-based implant.

Embodiment 1

In embodiment 1, a first metal selected from calcium and strontium is ion-implanted into the surface of a titanium-based implant as a base to form an inner layer, and an oxide of at least one second metal selected from magnesium, calcium, zinc, sodium, and potassium is deposited on the inner layer by a sputtering method to form an intermediate layer and an outer layer. Namely, the titanium-based implant surface composite coating is provided with an M-Ti-O inner layer formed by performing M ion implantation on the titanium-based implant surface, an M-N-Ti-O intermediate layer formed by depositing N oxide on the inner layer through a sputtering method, and an N oxide outer layer.

The titanium-based implant surface composite coating of the present embodiment is composed of, for example, a calcium ion-implanted layer (Ca-Ti-O layer, inner layer) formed by implanting calcium metal ions into the surface of the medical titanium-based implant, a Ca-Mg-Ti-O transition layer (intermediate layer) formed by depositing magnesium oxide (magnesium oxide) on the inner layer by sputtering, and a magnesium oxide (MgO) thin film (outer layer). In the medical titanium-based implant surface composite coating, calcium exists in the forms of calcium oxide and simple substance calcium, and magnesium exists in the form of magnesium oxide. Specifically, the calcium element in the inner layer exists in the form of calcium oxide and simple substance calcium, and further contains a simple substance titanium and titanium oxide. The calcium element in the intermediate layer exists in the form of calcium oxide and simple substance calcium, the magnesium element exists in the form of magnesium oxide, and titanium oxide (simple substance titanium and titanium oxide) can be further contained. When the contents of the M and N components are changed, the contents of titanium and titanium oxide are also changed, and the distribution of the titanium and titanium oxide in the intermediate layer can play a role in assisting the timing. The magnesium element in the outer layer exists in the form of magnesium oxide. The first and second metals are similar to each other when they are of other types described later. A coating containing Mg and Ca metal and oxides thereof is constructed on the surface of the titanium to generate an alkaline microenvironment containing Mg/Ca ions, so that the surface of the titanium can be endowed with antibacterial and bone-promoting properties. In addition, Mg and Ca are also essential macroelements for human body, and the biological safety can be effectively guaranteed.

The inner layer, the middle layer and the outer layer are sequentially arranged on the surface of the titanium-based implant. The outer layer is further from the substrate than the inner layer and the intermediate layer. The thickness of the second metal oxide film on the outer layer can be regulated and controlled, and the degradation time of the second metal oxide film is controlled, so that different requirements of different implantation operations on antibacterial time are met. For example, the degradation time of MgO is positively correlated with the thickness of MgO, and the antibacterial effect can be maintained by regulating the thickness of MgO film. The requirements for the antibacterial time are different because the wound areas of different implantation operations are different and the operation methods are different. Thus, according to different requirements, outer layers of different thicknesses can be prepared to meet the specific requirements. For example, in some orthopedic minimally invasive surgery, the wound surface is very small (the probability of contacting a bacterial environment is low, the risk of infection is low), and strong antibiosis of only a few hours or a few minutes is needed after implantation, so that the outer layer can be made very thin. In one example, the degradation of magnesium oxide in the outer layer may last up to 15 days from exposure to the physiological environment. In this embodiment, the thickness of the outer layer may be 1 to 1000 nm. In one example, the outer layer has a thickness of greater than 500nm, and the degradation of the second metal oxide in the outer layer may last for greater than about 14 days from exposure to the physiological environment. In addition, the second metal can also replace magnesium, one of calcium, zinc, sodium and potassium is adopted as the second metal, and oxides of magnesium, calcium, zinc, sodium and potassium generate strong alkaline microenvironment to effectively resist bacteria. An outer layer is disposed on a surface of the intermediate layer and has a composition comprising a second metal oxide. In this embodiment, the outer layer component is the second metal oxide.

And controlling the degradation time of the second metal oxide in the outer layer in the physiological environment by regulating the thickness of the outer layer film. In one embodiment, a radio frequency magnetron sputtering technology is adopted, a magnesium oxide target is selected, an MgO film is deposited on the acid-washed titanium surface, and the modified titanium surface (represented by MgO-Ti-1, MgO-Ti-2 and MgO-Ti-3 respectively) on which the MgO films with different thicknesses are deposited is obtained by controlling the magnetron sputtering technological parameters. The specific process conditions and parameters of magnetron sputtering and the sample names are shown in Table 1. The thickness of the obtained MgO-Ti modified titanium surface film is shown in FIG. 16.

TABLE 1

Sample numbering	MgO-Ti-1	MgO-Ti-2	MgO-Ti-3
				Air pressure	5.0×10 ^-1 Pa	5.0×10 ^-1 Pa	6.7×10 ^-1 Pa
Sputtering power	300W	300W	400W
				Time of sputtering	20min	40min	120min

FIG. 16 is a photograph showing the cross-sectional view of the magnesium oxide film on the surface of the MgO-Ti-1, MgO-Ti-2 and MgO-Ti-3 samples. As can be seen from the figure, the thicknesses of the MgO thin films on the surfaces of the MgO-Ti-1, MgO-Ti-2 and MgO-Ti-3 samples were 48.3nm, 98.7nm and 479.6nm, respectively.

The titanium material obtained through the modification treatment in the above embodiment is analyzed and detected for the surface ion release amount in physiological saline. The samples were placed in 15mL centrifuge tubes, 10mL saline was added, and soaked at 37 ℃ for 1,4, 7 and 14 days. The leachate was collected at each time point and 10mL of fresh saline was added again. The collected leachate was tested for Mg ion content by inductively coupled plasma atomic emission spectrometry (ICP-AES, Varian Liberty 150, USA).

FIG. 17 shows the results of Mg ion release after soaking samples of MgO-Ti-1, MgO-Ti-2 and MgO-Ti-3 in physiological saline for various periods of time. As can be seen, after 1 day of soaking, the MgO-Ti-1 and MgO-Ti-2 samples had substantially no release of Mg ions; after soaking for 1 day, the Mg ion release amount of the MgO-Ti-3 sample is gradually reduced, but the Mg ion release amount is still obvious, and the Mg ion release amount can exist for about 14 days; indicating that a MgO film having a thickness of 48.3nm or 98.7nm can exist in physiological saline for about 1 day; the MgO film having a thickness of 479.6nm can be present in physiological saline for about 14 days. The above results provide a reference for the selection of the initial antimicrobial coating thickness.

The Ca element can be implanted to a depth (thickness of the inner layer) of 1 to 150 nm. A shallower depth may be implanted as desired. The Ca atomic percentage of the modified titanium surface (Ca atomic percentage of the inner layer) may be 20% or less. In addition, the composite material can also replace calcium, the first metal adopts strontium, and the calcium, the strontium and oxides thereof generate a weak alkaline microenvironment in a physiological environment, and the biocompatibility is excellent. In one example, the first metal ions (e.g., Ca ions) on the modified titanium surface are released from a short time after the implantation begins and can be released continuously for 0-50 days. The "short period of time" is not particularly limited, and may be, for example, 0.5 hour depending on the actual condition. The first metal and titanium metal material mixture layer can be vertically oriented to the direction of the titanium metal material substrate, and the first metal is distributed on the surface of the substrate and in the shallow layer.

The thickness of the intermediate layer (in this embodiment, the Ca-Mg-Ti-O transition layer) may be 1 to 150 nm. In one example, the thickness of the intermediate layer is 10 to 150 nm. In another example, the thickness of the Ca-Mg-Ti-O transition layer is 50nm or more. In addition, strontium may be used as the first metal, or calcium, zinc, sodium, potassium may be used as the second metal. In one example, the middle layer may last for about seven days, and the inner layer may be present for at least 49 days.

In a preferable scheme, in the intermediate layer, the distribution of titanium element increases progressively along the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, the distribution of oxygen element increases progressively first and then decreases progressively along the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, the distribution of first metal element increases progressively along the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, and the distribution of second metal element decreases progressively along the thickness direction of the intermediate layer from the surface of the intermediate layer to the surface of the inner layer, so that the alkalinity of the surface microenvironment is further easy to change in a time sequence. This can be achieved by controlling specific process parameters such as the sequence and parameters of ion implantation, magnetron sputtering. For example, by changing the power in magnetron sputtering, the amount of argon plasma increases or decreases, and the amount of electrons generated, and the amount of target atoms/molecules generated by sputtering changes. The energy of different numbers of deposited particles is different, which affects the adsorption condition on the surface of the sample, and further affects the film forming condition. Eventually reflecting the difference in the distribution of the interlayer elements. For another example: parameters such as injection time, injection voltage and pulse width of M ion injection are changed, the number of M ions on the surface of the sample can be changed, and the injection depth of the M ions in the sample can be changed. In the field of solid surface modification, a significant difference between the crystal structure of the solid surface and the crystal structure of the solid interior is that the bonding chemical bond between atoms or molecules is interrupted, and the interrupted bond formed by the atoms or molecules on the solid surface is called a dangling bond, which has the ability to attract foreign atoms or molecules. By changing the parameters of M ion implantation, the components and the content of the surface of the sample can be changed, the number and the types of dangling bonds on the surface of the sample are changed, the combination between the surface of the sample and external atoms or molecules is influenced during magnetron sputtering, and different adsorption effects are generated. Specifically, the above changes affect, for example, the size, number, density and growth of crystal nuclei formed by deposition of the atoms/molecules of the magnetron target material on the surface of the sample, and further form the intermediate layer with different element distributions. The distribution rule of the elements in the middle layer in the embodiment can be effectively formed by regulating and controlling the M ion implantation process and the N oxide magnetron sputtering process.

Time-ordered services can be distributed through the elements of the middle layer. In an actual application scene, the middle layer is exposed to be in contact with a physiological environment after the outer layer is consumed to a certain degree. Can meet the special function requirement in the physiological environment through the designated rule of the element distribution. In particular, the period during and one week after surgery is the most susceptible to bacterial infection, and therefore the antibacterial effect during this period is particularly critical, and it is expected that the outer N-oxide will generate a strong alkaline microenvironment as soon as possible to meet the initial antibacterial demand. However, the composite film layer inevitably releases M ions at the same time in the initial stage, because M ions are released from the exposed part of the intermediate layer due to the difference in the rate of consumption of the N oxide layer at each part of the outer layer. In light of the above practical need, it was initially desired to achieve effective antibacterial purposes without the premature consumption of osteogenic M ions. In order to avoid premature consumption of the M ions, but in the face of the initial inevitable exposure of the intermediate layer to the physiological environment, it is preferred to choose a controlled release rate of the M ions. Slowing the release rate of the M ions avoids their premature consumption. As a method of slowing down the release rate of M ions, the element distribution of the intermediate layer is controlled. Preparing the intermediate layer with the specific element distribution rule. In the intermediate layer, the content of N element is decreased progressively, the content of M element is increased progressively, the transition from strong to weak of an alkaline microenvironment can be brought, and the damage to cells caused by sudden environmental change (the condition without the intermediate layer) is avoided. On the other hand, this trend of the content of the elements N and M in the intermediate layer allows a good balance between the antibacterial properties and the bone properties. Because the possibility of bacterial infection has been greatly reduced by the previously persistent strongly alkaline microenvironment in the middle post-operative period, e.g., after about 7 days post-operative, osteogenic properties are required to begin to function, but some antibacterial properties are also required to avoid bacterial entrapment. At this time, a smart transition of the strength of the alkaline microenvironment, i.e. the intermediate layer element distribution of the present invention, can play a role. In this distribution, it can also be seen that the titanium element increases and the oxygen element increases and decreases. The reasons and mechanisms for this distribution are as described above. The distribution also has the effects of slowing the release rate of M ions in the early stage and balancing the transition of strong and weak alkaline microenvironments in the middle stage. The oxygen element is increased and then decreased, the content of the oxygen element reaches the highest value at the middle position of the middle layer, at the position, the oxygen element exists in the form of N oxide, M oxide and titanium oxide, and as the stability of the active metal oxide is greater than that of a simple substance, when the film layer is consumed to the position, the oxide with better chemical stability is contacted with the physiological environment, and the purposes of slowing down the release of M ions and balancing a strong weak and alkaline microenvironment can be effectively achieved. By designing and preparing an intermediate layer with such an element distribution, the clinical requirements are further met: the primary antibacterial effect is mainly achieved in the early stage after the operation, the bone formation is promoted and continuously prevented in the middle stage of the operation, and the bone formation requirement is further improved in the later stage of the operation (about 20 days after the operation).

In one example, the initial period may be defined as the beginning of the operation to 7 days (1 week) after the operation; the middle stage is 7 days to 28 days (1 to 4 weeks), and the later stage is 28 days later. It can also be defined as early and late (before and after 1 week).

The titanium-based implant may be a titanium metal material, a titanium metal material having a titanium oxide coating (titanium oxide coating) on the surface thereof, or a titanium metal material having at least one of a micro-scale structure, a sub-micro-scale structure, and a nano-scale structure on the surface thereof. The titanium metal material can be pure titanium or titanium alloy.

The medical titanium surface composite coating (composite modified coating) according to the present embodiment is composed of a Ca — Ti — O layer (inner layer), a Ca — Mg — Ti — O layer (intermediate layer), and an MgO film (outer layer). Under physiological environment, the alkalinity of the composite coating surface microenvironment is changed in a time sequence, and a staged antibacterial osteogenesis effect can be generated. In the initial stage, the MgO film on the outer layer of the composite coating can quickly react with water to form a strong alkaline microenvironment so as to generate a good antibacterial effect and avoid bacterial infection in the initial stage of implantation; the middle layer can release calcium ions at the same time, and the osteogenic differentiation of the mesenchymal stem cells is promoted; along with the gradual degradation of the MgO film, the Ca-Ti-O layer in the later-stage composite coating can slowly react with water for a long time to form a weakly alkaline microenvironment containing Ca ions, inhibit the proliferation of bacteria, promote the adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells, and further realize good osseointegration.

The first metal can also be strontium, and the second metal can also be calcium, zinc, sodium or potassium. The medical titanium surface composite coating has the advantage that the alkalinity of the surface microenvironment of the medical titanium surface composite coating is changed in a time sequence manner under the physiological environment, and the staged antibacterial osteogenesis effect can be generated. In the initial stage, the second metal oxide (such as MgO) on the outer layer of the composite coating reacts with body fluid to generate a strong alkaline microenvironment which has strong antibacterial capacity and can kill bacteria attached to the surface of the composite coating and prevent the bacteria from colonizing the surface of the implant. As the reaction proceeds, the second metal oxide is consumed and the thickness decreases. Further, consumption of the outer second metal oxide exposes the intermediate layer, either partially or completely, to the physiological environment, and the intermediate layer begins to participate in the reaction of the physiological environment. The first metal ions in the middle layer are released, so that the biocompatibility of an alkaline microenvironment generated by the second metal oxide can be improved, and the adhesion, proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells are promoted. The mechanism is presumed to be as follows: the strong alkaline microenvironment causes the change of the internal pH of the cell protein and the change of the charged property of internal groups, so that the spatial structure of the protein is changed and even disintegrated, and a plurality of unfolded peptide chains are intertwined together to form obvious precipitates. The peptide bond is hydrolysed under base catalysis, resulting in a shortening of the peptide chain, which in turn leads to the gradual disappearance of the previously produced precipitate. The intermediate layer releases first metal ions (calcium ions and strontium ions with the same electron number and ion charge quantity in the outermost layer), and on one hand, the first metal ions can form first metal hydroxide with low water solubility in an alkaline microenvironment, so that the alkalinity is reduced to a certain extent, and the damage to cell proteins is reduced. On the other hand, the first metal ion can be combined with the protein on the cell surface to achieve charge balance, so that the protein combined with the first metal ion can resist the change of the group charge property to a certain extent. After the intermediate layer is consumed, the inner layer reacts with a physiological environment to form a weak alkaline microenvironment, inhibits the proliferation of bacteria, continuously and slowly releases first metal ions (including the surface of the substrate and the shallow layer embedded in the substrate), promotes the adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and finally realizes good osseointegration. In addition, in the present embodiment, the case where the inner layer is formed by performing the ion implantation of the first metal on the surface of the titanium-based implant and the intermediate layer and the outer layer are formed by depositing the oxide of the second metal on the inner layer by the sputtering method is described, but the processes for preparing the inner layer, the intermediate layer and the outer layer of the composite coating on the surface of the titanium-based implant of the present invention are not limited to these methods, and the structure of the composite coating on the surface of the titanium-based implant of the present invention may be formed by using a known preparation process.

Embodiment 2.

The surface modification method of the titanium-based implant (hereinafter sometimes referred to as titanium-based material) based on the titanium metal material comprises the steps of firstly adopting a plasma immersion ion implantation technology to implant first metal ions into the surface of the titanium-based material to construct and obtain an inner layer; and then depositing a second metal oxide on the surface of the inner layer by adopting a magnetron sputtering technology, and further constructing to obtain an intermediate layer and an outer layer. The following exemplary description describes the surface modification method of titanium-based materials according to the present disclosure, and typically describes the method for forming the medical titanium surface composite coating according to embodiment 1 above by surface modification of a titanium-based implant, but is not limited thereto.

Firstly, first metal ions such as calcium ions are implanted on the surface of the medical titanium-based material by adopting plasma immersion ion implantation. As the titanium-based material, pure titanium, a titanium alloy, or a titanium oxide coating may be used. The titanium-based implant may be subjected to grinding, turning, etching, ultrasonic cleaning, or additive manufacturing techniques such as 3D printing, contouring, stereolithography, prior to implantation. The surface of the titanium-based implant may also be pretreated prior to implantation, including polishing and cleaning, or polishing and cleaning followed by grit blasting. In some embodiments, the pretreatment may be a titanium-based material surface polishing, cleaning, drying, or the like. As an example, a 10mm by 1mm pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water in this order for 10 minutes each, and then the surface was ultrasonically acid-washed with a mixed acid wash (made by mixing hydrofluoric acid, nitric acid and ultrapure water in a volume ratio of 1:5: 4), followed by ultrasonic cleaning with deionized water to obtain a clean rough acid-washed titanium surface.

A first elemental metal or a first metal-Ti alloy (e.g., pure calcium) can be used as the cathode. And injecting calcium ions into the surface of the pretreated titanium-based material by adopting a plasma immersion ion injection technology to obtain the calcium ion injection layer. Plasma immersion ion implantation/coating (PIII)&D) The technical process parameters can be as follows: setting the temperature of a vacuum chamber for plasma immersion ion implantation to be 10-100 ℃, and the background vacuum degree to be 1 multiplied by 10 ^-3 ～6×10 ^-3 Pa, the injection voltage is-5 to-50 kV, the frequency is 5 to 20Hz, the pulse width is 100 to 1000 mus, and the injection time is 0.5 to 240 minutes. The preferred range is that the temperature of the vacuum chamber is 20-50 ℃, and the background vacuum degree is 3 multiplied by 10 ^-3 ～5×10 ^-3 Pa, the injection voltage is-20 to-40 kV, the frequency is 7 to 15Hz, the pulse width is 400 to 600 mus, and the injection time is 90 to 180 minutes. The invention adopts plasma immersion ion implantation to implant calcium ions on the surface of the medical titanium-based material for modification, and can promote the proliferation of osteoblast-related cells by the medical titanium-based materialAnd up-regulates the expression of osteogenic and angiogenetic genes.

Then, a second metal material such as magnesium metal or magnesium oxide such as ceramic magnesium oxide is used as the target. A layer of MgO film can be deposited on the surface of the calcium ion injection layer by adopting a magnetron sputtering technology, and then the composite modified coating consisting of the Ca-Ti-O layer (inner layer), the Ca-Mg-Ti-O layer (middle layer) and the MgO film (outer layer) is constructed on the surface of the medical titanium. In one aspect of the disclosure, magnetron sputtering is performed with ceramic magnesium oxide as a target, and only argon is introduced during the magnetron sputtering process. In another aspect of the disclosure, the magnetron sputtering is performed using magnesium metal as a target, and argon and oxygen are simultaneously introduced during the magnetron sputtering. The technological parameters of the magnetron sputtering technology can be as follows: applying radio frequency magnetron sputtering on the target material, setting the temperature of a vacuum chamber for magnetron sputtering to be 10-100 ℃, and the background vacuum degree to be 1 multiplied by 10 ^-3 ～6×10 ^-3 Pa, radio frequency power of 100-600W, working pressure of 1 × 10 ^-1 20Pa and the sputtering time is 30-240 minutes. The preferable range is that the temperature of the vacuum chamber is 20-50 ℃, and the background vacuum degree is 3 multiplied by 10 ^-3 ～5×10 ^-3 Pa, radio frequency power 300-500W, working pressure 3X 10 ^-1 5Pa, and 60 to 180 minutes of sputtering time. In one example, radio frequency magnetron sputtering is applied to a magnesium oxide target, the temperature of a vacuum chamber of the magnetron sputtering is set to be 20-50 ℃, and the background vacuum degree is 3 multiplied by 10 ^-3 ～5×10 ^-3 Pa, radio frequency power of 300-500W, Ar gas flow of 1-50 sccm, and working pressure of 3 × 10 ^-1 5Pa, and the sputtering time is 60-180 minutes; or applying radio frequency magnetron sputtering on the metal magnesium target material, wherein the temperature of the vacuum chamber is 20-50 ℃, and the background vacuum degree is 3 multiplied by 10 ^-3 ～5×10 ^-3 Pa, radio frequency power of 300-500W, Ar gas flow of 1-50 sccm, O ₂ The flow rate is 1-50 sccm and the working pressure is 3 x 10 ^-1 5Pa, and 60 to 180 minutes of sputtering time. As an example, the ceramic magnesium oxide is used as a target material and the radio frequency magnetron sputtering technology is adopted (the technological parameters are that the flow rate of Ar gas is 30sccm, and the gas pressure is 6.7 multiplied by 10) ^-1 Pa, sputtering power of 400W and sputtering time of 120min) to deposit MgO on the surface of the calcium ion injection modified titanium material, thus obtaining the composite modified titanium material.

According to the method of the embodiment, a composite coating consisting of a magnesium oxide layer, a magnesium calcium oxide titanium layer and a calcium titanium layer is constructed on the surface of titanium metal by combining a plasma immersion ion implantation technology and a magnetron sputtering technology. A Ca-Ti layer is constructed on the surface of titanium by adopting a plasma immersion ion injection technology, so that calcium ions are slowly reacted with water to release, a weakly alkaline surface microenvironment is generated, and the adhesion and proliferation of mesenchymal stem cells and the expression of osteogenic genes can be promoted in a long time range. In the embodiment, a layer of MgO film and a layer of Ca-Mg-Ti-O are constructed outside a Ca-Ti layer on the surface of titanium modified by injecting calcium ions by adopting a magnetron sputtering technology, so that the composite modified titanium can react with water under a physiological environment at the initial stage to quickly generate hydroxyl and magnesium ions, a strong alkaline microenvironment is formed, and a good antibacterial effect is generated; meanwhile, calcium ions are released to improve the biocompatibility of the microenvironment and promote the osteogenic differentiation of the bone marrow mesenchymal stem cells. The degradation time of MgO is in positive correlation with the thickness of MgO, and the antibacterial effect of MgO can be maintained in a certain thickness range through regulation. Therefore, the thickness of the titanium surface can be controlled by regulating and controlling the magnetron sputtering process parameters, so that the antibacterial property of the modified titanium surface can be ensured within a time range with specific requirements, and the clinical requirements can be met.

And the modified titanium material obtained by modification obtains good osteogenic effect, and simultaneously obtains good antibacterial effect by constructing an alkaline coating through biologically safe Mg and Ca elements, thereby avoiding the adverse effect of common antibacterial components on the cell osteogenic activity. And the thickness of the MgO film and the doping depth and content of Ca can be controlled by adjusting the technological parameters of magnetron sputtering and ion implantation, so that the alkalinity and the duration of the microenvironment on the surface can be regulated and controlled, the actual clinical requirement can be met, and the problems of insufficient antibacterial performance and osseointegration capability of the titanium implant in clinic at present can be solved.

The surface modification method for the titanium-based orthopedic implant and the composite coating on the surface of the titanium-based orthopedic implant have the antibacterial effect that the basic microenvironment (namely hydroxide) on the local surface generated based on the modified coating in the physiological environment consumes ' the proton coupling respiration electron transfer chain outside the bacteria ' to obstruct the energy metabolism of the bacteria ' and induce the bacteria to generate oxidative stress to realize the antibacterial effect. The invention has the time sequence function aiming at the surgical implantation operation, namely, the invention plays different roles aiming at different operation stages, and finally achieves more excellent antibacterial and osseointegration effects. The coating structure of the invention comprises an inner layer, a middle layer and an outer layer. For example, when the first metal is calcium and the second metal is magnesium, the inner layer: the chemical composition comprises three elements of titanium, calcium and oxygen. Calcium, which contributes to the bone function, is embedded in the titanium surface layer, i.e. present at the surface and in the shallower interior of the titanium substrate, in a discontinuous but uniformly distributed form at the surface and at a depth of about tens of nanometers in the shallower interior. And thus may also be described as a mixture of the first metal and titanium. Outer layer: the magnesium oxide is magnesium oxide with antibacterial function, and continuously and completely covers the outermost layer. An intermediate layer: the transition layer is arranged between the outer layer and the inner layer. In chemical composition, titanium, calcium, oxygen, magnesium are included, i.e. all elements comprising an inner layer and an outer layer. The element distribution of the middle layer has a certain rule in the vertical direction of the thickness: the distribution of titanium element increases progressively from the surface of the intermediate layer to the surface of the inner layer along the vertical direction (the thickness direction of the intermediate layer), the distribution of oxygen element increases progressively from the surface of the intermediate layer to the surface of the inner layer along the vertical direction first and then decreases progressively, the distribution of calcium element (first metal element) increases progressively from the surface of the intermediate layer to the surface of the inner layer along the vertical direction, and the distribution of magnesium element (second metal element) decreases progressively from the surface of the intermediate layer to the surface of the inner layer along the vertical direction. In an application scene, the titanium implant with the coating is implanted into a human body along with the progress of a surgical operation, and only the outer layer is in contact with a physiological environment such as body fluid in the initial stage (within about 1 day after implantation), so that the outer layer starts to play a role first. The magnesium oxide on the outer layer reacts with body fluid to generate a strong alkaline microenvironment on the surface of the magnesium oxide, and the strong alkaline microenvironment has strong antibacterial capacity and can kill bacteria attached to the surface of the magnesium oxide and prevent the bacteria from colonizing the surface of the implant. In the actual operation process, if the propagation and the existence of bacteria are prevented in the early stage of the operation, the infection probability after the operation can be greatly reduced. Thus, the period during and 24 hours after surgery is a critical period for antisepsis. The strong alkaline microenvironment of the outer magnesium oxide layer can ensure the excellent antibacterial effect in the early stage after the operation. At the same time, the alkalescence of the microenvironment can produce negative effects on cells, such as inhibiting the adhesion and proliferation of cells, but promoting the osteogenic differentiation. As the reaction proceeds, magnesium oxide is continuously consumed and the thickness decreases. The consumption of the outer magnesium oxide exposes the intermediate layer, either partially or completely, to the physiological environment, and the intermediate layer begins to participate in the reaction of the physiological environment. The calcium ions in the middle layer are released, so that the biocompatibility of an alkaline microenvironment generated by magnesium oxide can be improved, and the adhesion, proliferation and osteogenic differentiation of the mesenchymal stem cells are promoted. Calcium ions can reduce the negative effects of the alkaline microenvironment on cells to a certain extent. With the consumption of the outer layer and the middle layer, the alkaline microenvironment gradually changes from strong to weak to an environment more suitable for cell growth and adhesion, and the growth and healing of bone tissues are promoted. This phase occurs for a period of time (e.g., 1-50 days) after the implant enters the body, corresponding to exactly the time for bone tissue to heal and grow. The promotion of calcium ions enables bone tissues to be more easily healed and grown, and can effectively shorten the osseointegration time. After the middle layer is consumed, the inner layer reacts with a physiological environment to form a weak alkaline microenvironment, inhibits the proliferation of bacteria, continuously and slowly releases calcium ions (including the surface of the substrate and the shallow layer embedded in the substrate), promotes the adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and finally realizes good osseointegration. The alkalescent microenvironment generated by the inner layer has certain bacteriostatic ability, and is functionally a continuation of the antibacterial ability of the alkalescent microenvironment at the initial stage after the operation, thereby reducing the infection risk. The promotion of the alkalescent microenvironment containing calcium ions to bone tissue repair can effectively improve the osseointegration effect. The coating has the characteristic of time sequence, namely, the coating has special function correspondence aiming at the early stage of operation, the post-operation and the post-operation recovery period. Strong antibacterial property is generated by a strong alkaline microenvironment in and at the initial stage of operation, so that the infection risk after operation is greatly reduced; in the postoperative recovery period, the alkaline microenvironment is weakened from strong, so that the growth of bone tissues is promoted, the osseointegration speed is accelerated, and the osseointegration effect is improved. The bacteriostatic ability of the alkalescent microenvironment ensures effective avoidance of postoperative infection.

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 of the following examples;

in the following examples, reagents, materials and instruments used are all conventional reagents, conventional materials and conventional instruments, which are commercially available, if not specifically mentioned, and the reagents involved therein can also be synthesized by conventional synthesis methods.

Example 1

(1) Ultrasonically cleaning a pure titanium metal sheet with the thickness of 10mm multiplied by 1mm by using acetone, absolute ethyl alcohol and deionized water in sequence for 10 minutes each time, and then carrying out ultrasonic pickling treatment, wherein a pickling solution is formed by mixing hydrofluoric acid, nitric acid and ultrapure water according to a volume ratio of 1:5: 4; followed by ultrasonic cleaning with deionized water to obtain a clean and uniform surface, i.e., a pickled titanium surface (denoted as Ti). The resulting surface was a rough microstructure, and protrusions were found on the surface under high power mirrors (see FIG. 1).

(2) And (3) adopting a plasma immersion ion implantation technology, selecting a metal calcium cathode, performing calcium ion implantation on the surface of the titanium obtained by cleaning, and constructing a Ca-Ti layer on the surface of the titanium. Specific process conditions and parameters are shown in table 2. The surface topography of the modified titanium sample (denoted Ca-Ti) obtained under this process parameter is shown in fig. 1. The results show that after the calcium ion implantation, the original convex structure has basically disappeared, and the surface of the sample becomes flatter, which is caused by the calcium ion bombardment effect.

Table 2 ion implantation process conditions and parameters

Background vacuum	4.0×10 ^-3 Pa
		Injection voltage	﹣30kV
Pulse width	500μs
		Frequency of pulses	10Hz
Injection time	120min

(3) The method adopts a radio frequency magnetron sputtering technology, selects a magnesium oxide target material, and deposits a MgO film on the surface of calcium ion injection titanium to obtain a composite modified titanium surface (represented by MgO @ Ca-Ti) with the surface composed of a Ca-Ti inner layer, a Ca-Mg-Ti-O intermediate layer and a MgO film outer layer. For comparison, a titanium pickled surface (represented by MgO-Ti) modified by magnetron sputtering only was prepared. Specific process conditions and parameters of magnetron sputtering are shown in Table 3;

TABLE 3

Air pressure	6.7×10 ^-1 Pa
		Sputtering power	400W
Time of sputtering	120min

The surface appearance of the obtained MgO-Ti modified titanium is shown in figure 1, and the surface appearance of a MgO-Ti sample obtained by modifying acid-washed titanium through MgO magnetron sputtering under a low power lens is similar to that of the acid-washed titanium and presents a micron structure. The surface can be clearly observed to be uniformly distributed with nano particles under a high power lens, and a convex structure still exists. The surface appearance of the composite modified titanium MgO @ Ca-Ti sample is shown in figure 1, the surface has no obvious convex structure, but uniformly distributed nano particles can be observed. Fig. 2 is an XRD spectrum of the single calcium ion-implanted modified titanium, the single magnetron sputtering modified titanium sample, the composite modified titanium sample and the unmodified titanium sample obtained by the modification treatment of this example. As can be seen from FIG. 2, the calcium ion-implanted modified titanium sample only detected the characteristic peak of metallic titanium, indicating that no new phase was generated in the modified layer; and the characteristic peak of magnesium oxide is detected by the magnetron sputtering modified titanium and the composite modified titanium, which indicates that the surface of the titanium is covered with a layer of magnesium oxide film.

FIG. 3 is the XPS survey spectra of single calcium ion-implanted modified titanium, single magnetron sputtering modified titanium, composite modified titanium and unmodified titanium samples obtained by the modification treatment of this example, and the high resolution spectra of Ca on the surface of Ca-Ti sample, MgO-Ti sample surface Mg, and MgO @ Ca-Ti sample surface Mg. As can be seen from fig. 3, calcium exists in the form of calcium oxide and elemental calcium on the surface of the modified titanium sample by calcium ion implantation, while Mg exists mainly in the form of MgO on the surface of the titanium-based material obtained by the modification treatment.

Fig. 4 shows the results of the depth distribution of surface elements of the single calcium ion implantation modified titanium, the single magnetron sputtering modified titanium sample, and the composite modified titanium sample obtained by the modification treatment of this example. As can be seen from FIG. 4, the depth of the calcium ion implantation in the resulting modified titanium surface layer was 150nm, the thickness of the Ca-Mg-Ti-O transition layer was 100nm, and the thickness of the MgO film was about 750 nm.

Example 2

The titanium material obtained by the modification treatment of the above example 1 was analyzed and examined for the amount of surface ion release in physiological saline. The samples were placed in 15mL centrifuge tubes, 10mL physiological saline was added, and soaked at 37 ℃ for 1,4, 7, 14, 21, 35, and 49 days. The leachate was collected at each time point and 10mL of fresh saline was added again. The collected leachate was tested for Mg and Ca ion content by inductively coupled plasma atomic emission spectrometry (ICP-AES, Varian Liberty 150, USA).

Fig. 5 shows the release results of Mg ions (5 a in fig. 5) and Ca ions (5 b in fig. 5) after the single calcium ion-implanted modified titanium, the single magnetron sputtering modified titanium sample, and the composite modified titanium sample obtained by the modification treatment of example 1 are soaked in physiological saline for different periods of time. According to 5a, after the sample is soaked for 14 days, the release of Mg ions of the MgO-Ti sample and the MgO @ Ca-Ti sample is obviously reduced, which indicates that the MgO film on the surface of the composite modified titanium sample can exist in normal saline for about 14 days; in addition, Mg ion release rate was fast in the initial 1,4 and 7 days. From 5b, Ca-Ti and MgO @ Ca-Ti samples release Ca ions within 49 days, which shows that the Ca-Ti layer on the surface of the composite modified titanium sample can continuously and slowly release the Ca ions.

Example 3

The titanium material obtained by the modification treatment in example 1 was evaluated for the degree of alkalinity of the local surface microenvironment in physiological saline. The sample was placed in a 15mL centrifuge tube, 5mL of physiological saline was added, and the tube was soaked at 37 ℃ for 24 hours. Using a pH meter (FE 20-FiveEasy) ^TM METTLER TOLEDO) the pH of the saline at each time point was measured.

The pH of the physiological saline after soaking the Ti, Ca-Ti, MgO-Ti and MgO @ Ca-Ti samples for 24 hours is shown in FIG. 6. The pH values of the normal saline of the Ti and Ca-Ti sample groups are similar, while the pH values of the normal saline of the MgO-Ti and MgO @ Ca-Ti sample groups are both over 10 and are obviously greater than that of the Ti sample group, which indicates that the surface of the MgO film can quickly form an alkaline microenvironment.

Example 4

An antibacterial experiment was performed on the material prepared by example 1, using the titanium obtained by the acid cleaning treatment in example 1 as a control (denoted as Ti): sterilizing all samples with 75% alcohol solution, drying, and adding 10% ethanol ⁷ The cfu/mL bacterial solution was dropped on the surface of the sterilized sample (60. mu.L/cm) ² ) Then, the sample with the bacterial liquid is put into a constant temperature incubator at 37 ℃ for 24 hours. Taken out for 24h cultureThe sample is fixed by glutaraldehyde solution with volume fraction of 2.5%, then dehydrated and dried by gradient alcohol solution and hexamethyldisilazane solution, and observed under a scanning electron microscope, and the result is shown in fig. 7 (row 1). Meanwhile, LIVE-DEAD staining was performed on bacteria on the surface of the sample using LIVE/DEAD Bac Light kit (L13152). The bacteria were inoculated on the surface of the sample and cultured for 24 hours, and the sample was rinsed twice with 800. mu.L of physiological saline. Subsequently, 500. mu.L of a dye was added to each sample well, staining was performed at room temperature for 15min, the sample was rinsed 2 times with 800. mu.L of physiological saline, and the stained bacteria on the surface of the sample were observed by a fluorescence microscope, as shown in FIG. 7 (

rows

2,3, and 4).

As can be seen from FIG. 7, a large number of Staphylococcus aureus can be observed on the surface of the Ti sample, and the bacteria form is complete and not damaged; compared with the surface of a titanium pickling sample, the number of bacteria on the surface of the Ca-Ti sample is slightly reduced, but the shape of the bacteria is also in an integral state and has no obvious damage; the number of staphylococcus aureus on the surfaces of the MgO-Ti and MgO @ Ca-Ti samples is obviously reduced, and part of bacteria is damaged, so that the MgO film can effectively kill the staphylococcus aureus attached to the surfaces.

As can be seen from FIG. 7, a large number of live Staphylococcus aureus could be observed on the Ti sample surface, and a small number of dead bacteria were observed, indicating that the bacteria grew well; compared with Ti, the number of the staphylococcus aureus on the surface of the Ca-Ti sample is reduced, but the staphylococcus aureus is still live bacteria, which indicates that the bacteriostatic effect is weaker; the number of the staphylococcus aureus on the surfaces of the MgO-Ti and MgO @ Ca-Ti samples is obviously reduced, and a plurality of dead bacteria can be observed, which shows that the MgO film can effectively kill the staphylococcus aureus and has good antibacterial effect.

Example 5

An antibacterial experiment was performed on the material prepared by example 1, using the titanium obtained by the acid cleaning treatment in example 1 as a control (denoted as Ti): sterilizing all samples with 75% alcohol solution, drying, and respectively adding 10% ethanol solution ⁷ cfu/mL (high concentration) and 10 ⁶ cfu/mL (low concentration) of bacteria dropped on the surface of the sterilized sample (60. mu.L/cm) ² ) Then putting the sample with the bacterium liquid into the sampleCulturing at 37 deg.C for 24 hr. Then washing the bacterial liquid on the surface of the sample by using normal saline, and respectively diluting by 10 times, 100 times and 1000 times; then, 100. mu.l of each diluted bacterial solution was placed on nutrient agar plates, and the mixture was uniformly pushed with a glass rod (three plates for each diluted bacterial solution), and cultured in a 37 ℃ incubator for 24 hours, and then the number of colonies on the agar plates was observed, and the results are shown in FIG. 8 (high concentration inoculated bacteria) and FIG. 9 (low concentration inoculated bacteria).

As can be seen in FIG. 8, the inoculated bacteria were 10 ⁷ At cfu/mL, numerous colonies of Staphylococcus aureus were present on the agar plate surface of the Ti and Ca-Ti samples, and only single-digit bacterial colonies were observed with the agar plates of the MgO-Ti and MgO @ Ca-Ti samples, and the bacteria were essentially killed. The MgO film can generate good antibacterial action on high-concentration bacteria at the initial stage, and Ca-Ti basically has no obvious antibacterial action.

As can be seen from FIG. 9, the inoculated bacteria were 10 ⁶ When cfu/mL is obtained, the colony number of the agar plate of the Ca-Ti sample is obviously reduced compared with that of the Ti sample, and the result shows that the Ca-Ti sample has a certain antibacterial effect on low-concentration bacteria. No colony was found on the surface of the agar plates of the MgO-Ti and MgO @ Ca-Ti samples, indicating that all the bacteria were killed, indicating that the MgO film initially had a good antibacterial effect on the bacteria.

Example 6

The acid-washed titanium (Ti) and composite modified titanium (MgO @ Ca-Ti) materials prepared in example 1 were subjected to an antibacterial test to evaluate changes in the antibacterial properties of the surfaces of the samples after the materials were soaked in normal saline for various periods of time. After soaking the Ti and MgO @ Ca-Ti samples in 5ml of physiological saline at 37 ℃ for 1,4 and 7 days, the samples were sterilized with an alcohol solution having a volume fraction of 75%. After the samples were dried, the concentrations were 10 respectively ⁷ The cfu/mL bacterial solution was dropped on the surface of the sterilized sample (60. mu.L/cm) ² ) Then, the sample with the bacterial liquid is put into a constant temperature incubator at 37 ℃ for 24 hours. Then washing the bacterial liquid on the surface of the sample by using normal saline, and respectively diluting by 10 times, 100 times and 1000 times; then 100. mu.l of the diluted bacterial solutions were placed on nutrient agar plates, pushed uniformly by glass rods (three agar plates for each dilution factor bacterial solution), incubated at 37 ℃ for 24 hours, and the number of colonies on the agar plates was observed, as a resultAs shown in fig. 10.

As can be seen from FIG. 10, the colony count of Staphylococcus aureus on the surface of the agar plate of the MgO @ Ca-Ti samples was significantly less than that of the Ti sample group after soaking for 1 day and 4 days, and the antibacterial ratio to Staphylococcus aureus was 95.67% and 94.84%, respectively. After soaking for 7 days, the colony number of staphylococcus aureus on the surface of the agar plate of the MgO @ Ca-Ti sample is increased, but is still less than that of the Ti sample group, the antibacterial rate is 62.3 +/-5.3%, and certain antibacterial performance is still shown.

The results show that the Ca-Ti sample has no obvious antibacterial effect and can only inhibit the growth of bacteria under the condition of high-concentration bacteria; and the MgO-Ti and MgO @ Ca-Ti samples can maintain good antibacterial effect on staphylococcus aureus for at least 7 days.

Example 7

The samples prepared by example 1 were tested for cell adhesion performance. After sterilization of the sample, the density was 1.0X 10 ⁴ cells/mL mouse bone marrow mesenchymal stem cells (BMSCs) were seeded on the surface of sterilized samples and placed in CO ₂ Incubators were incubated at 37 ℃ for 1,4 and 24 hours. The cells were rinsed 1 time with 1mL PBS and fixed with 1mL 10% paraformaldehyde solution for 30 min. Microtubules, actins and nuclei of cells were stained with β -tubulin antibody, FITC-phalloidin and Hoechst, respectively, and the stained cytoskeleton was observed with a confocal laser fluorescence microscope and photographed, and the results are shown in fig. 11.

FIG. 11 is a photograph of skeletal staining of BMSCs cells cultured on the surface of Ti, Ca-Ti, MgO-Ti and MgO @ Ca-Ti samples for 1,4 and 24 hours. After the culture is carried out for 1 hour, most cells on the surface of the Ti sample are circular, and part of cells are in a polygonal form and have a tendency of spreading to the periphery; cells on the surface of the Ca-Ti sample already show a spindle shape, and the actin expression amount of the cells is obviously increased, which shows that the surface of the Ca-Ti sample can promote the initial adhesion of the mesenchymal stem cells; almost all cells on the surface of the MgO-Ti sample are circular, and the spreading area of the cells is small, so that the MgO-Ti sample has a strong inhibiting effect on the initial adhesion of the cells; the overall spreading area of the surface cells of the MgO @ Ca-Ti sample was also smaller than that of the Ti surface cells, but increased compared to that of the MgO-Ti sample, indicating that the biocompatibility of the MgO @ Ca-Ti sample was better than that of the MgO-Ti sample. After 4 hours of culture, the expression of cell actin and tubulin can be obviously observed on the surfaces of the Ti and Ca-Ti samples. Cells on the surface of the Ca-Ti sample are all spread; the cell spreading condition of the surface of the MgO-Ti sample is not obviously changed compared with that of the sample at the 1 st hour; while the cell portion on the surface of the MgO @ Ca-Ti sample had spread. After the sample is cultured for 24 hours, the cells on the surfaces of all samples are completely spread except that a small part of the cells on the surfaces of the MgO-Ti samples are still round, and have more actin and tubulin expressions, and abundant filamentous pseudopoda and platy pseudopoda of the cells can be observed, which indicates that the adhesion condition of the cells on the surfaces of the samples is good. The initial spreading area of the mesenchymal stem cells on the surface of each group of samples has the following rule: Ca-Ti, MgO @ Ca-Ti, MgO-Ti. The results show that the Ca-Ti sample is more beneficial to the initial adhesion of the mesenchymal stem cells of the bone marrow, which is probably caused by weak surface microenvironment alkalinity and the released Ca ions having the function of promoting cell adhesion; the MgO-Ti sample can inhibit the adhesion of cells at the initial stage, which is probably caused by strong microenvironment alkalinity of the surface of the MgO film; as for the MgO @ Ca-Ti sample, although the surface microenvironment is stronger in alkalinity, the adhesion condition of cells on the surface is better than that of the MgO-Ti sample due to the release of Ca ions.

Example 8

And (3) detecting the shapes of the scanning electron microscope of the surface cells of the titanium-based material and the pure titanium sample obtained by the modification treatment in the embodiment 1. After sterilization of the sample, the density was 5X 10 ⁴ 1ml of cell suspension per ml is inoculated on the surface of a sample, after being cultured for 7 days, the sample is washed 2 times by PBS, the sample is fixed by glutaraldehyde solution with the volume fraction of 2.5%, and then dehydrated and dried by gradient alcohol solution and hexamethyldisilazane solution, and the result is observed under a scanning electron microscope, and the result is shown in FIG. 12.

As can be seen from FIG. 12, both the Ti and Ca-Ti samples had substantially confluent surface cells and had good cell adhesion; only a small part of the area on the surface of the MgO-Ti sample is not covered by the cells, and part of the cells are not completely spread and are spherical; the number of cells on the surface of the MgO @ Ca-Ti sample is slightly less than that of the Ti and Ca-Ti samples, but the cells are completely spread, and the overall adhesion condition of the cells is better than that of the MgO-Ti sample.

Example 9

And (3) detecting the expression of the osteogenesis related genes of the cells on the surfaces of the titanium-based material and the pure titanium sample obtained by the modification treatment in the example 1. After sterilization of the sample, the density was 1X 10 ⁴ 1ml of the cell suspension per ml was inoculated on the surface of the sample, cultured for 7 days and 14 days, and the sample was transferred to a new well plate, and 1ml of Trizol reagent was added per well to extract intracellular RNA. cDNA (complementary DNA) was synthesized by reverse transcription using a First Strand cDNA Synthesis Kit (Transcriptor First Strand cDNA Synthesis Kit). The real-time quantitative PCR method was used to detect the expression of ALP, Osteopontin (OPN), osteogenic transcription factor Osterix (OSX) and Runx2 genes, and the results are shown in FIG. 13.

As can be seen from FIG. 13, the Ca-Ti, MgO-Ti and MgO @ Ca-Ti samples can promote the expression of the gene Runx2, which is the osteogenesis related transcription factor, and the effect of the Ca-Ti samples is particularly obvious. The Runx2 gene is a specific gene for osteogenic differentiation of bone marrow mesenchymal stem cells, can promote early immature differentiation of osteoblasts, and is also an upstream gene for osteoblast specific transcription factor (OSX), Osteopontin (OPN) and alkaline phosphatase (ALP). The Ca-Ti sample can remarkably promote the expression of Runx2 gene of the stem cell, further up-regulate the expression of bone-related genes of OSX, OPN and ALP at the downstream of the Runx2 gene, and promote the bone-forming differentiation of the stem cell. The promoting effect of the MgO-Ti and MgO @ Ca-Ti samples was not as good as that of the Ca-Ti samples, but was significantly higher than that of the Ti samples. The results show that the Ca-Ti, MgO-Ti and MgO @ Ca-Ti samples can obviously promote osteogenic differentiation of bone marrow mesenchymal stem cells, and the promotion effect shows the following rule: Ca-Ti > MgO @ Ca-Ti > MgO-Ti.

Example 10

The selective antibacterial effect of the titanium-based material and the pure titanium sample obtained by the modification treatment in example 1 on bacteria was evaluated by a co-culture experiment of bacteria and cells. 20 μ L of a bacterial suspension of E.coli and S.aureus (bacterial concentration 10) ⁴ cfu/mL) was inoculated to the sample surface. Incubate at 37 ℃ for 2 hours and wash the sample 3 times with 37 ℃ pre-warmed PBS. 1mL of a solution having a density of 5.0X 10 ⁴ Osteoblasts in cell/mL were seeded onto the sample surface. The cell-bacterial co-culture was performed with α -MEM cell culture medium without antibiotic addition but with 2% LB or NB bacterial culture medium. Culturing for 2 days, observing SEM appearances of cells and bacteria on the surface of the sample, washing the sample for 2 times by PBS, fixing the sample by glutaraldehyde solution with the volume fraction of 2.5%, then dehydrating and drying by gradient alcohol solution and hexamethyldisilazane solution, and observing under a scanning electron microscope, wherein the result is shown in figure 14. In addition, cells were stained with calcein and propidium iodide alive and observed by a fluorescence microscope, and the results are shown in fig. 15.

As can be seen from FIG. 14, when bacteria and cells were present, the surfaces of the experimental group Ti and Ca-Ti samples had been covered with a number of Staphylococcus aureus, had formed a pellicle, and no osteoblasts were found; no staphylococcus aureus is found on the surface of the MgO-Ti sample, and obvious osteoblasts can be observed; no bacteria are found on the surface of the MgO @ Ca-Ti sample, the osteogenic quantity is obviously increased compared with the surfaces of the Ti, Ca-Ti and MgO-Ti samples, and the cell adhesion spreading area is larger, which indicates that the cell growth state is good. As can be seen from FIG. 15, the number of live cells was small and the proportion of dead cells was large on the surface of the samples of Ti and Ca-Ti in the presence of bacteria. For the MgO-Ti sample group, the number of living cells was significantly increased, and the number of dead cells was also smaller. For the MgO @ Ca-Ti sample group, the number of viable cells was further increased, and there were no dead bacteria. The results show that the MgO-Ti and MgO @ Ca-Ti samples can generate good selective antibacterial effect in the initial stage of the surface, and the MgO @ Ca-Ti samples can release Ca ions in the initial stage of the surface, so that the cell compatibility is improved, and the MgO @ Ca-Ti samples have better selective antibacterial and cell adhesion promoting properties.

The results show that the titanium surface obtained by the composite modification of calcium ion implantation and magnesium oxide magnetron sputtering has good selective antibacterial osteogenesis effect, and can meet the requirements of better antibacterial performance required by the medical titanium implant material in the initial stage and better bone promoting performance required by the medical titanium implant material in the later stage.

Claims

1. The titanium-based implant surface composite coating is characterized by comprising an inner layer, a middle layer and an outer layer which are sequentially formed on a titanium-based implant substrate; the inner layer is formed inwards from the surface of the titanium-based implant along the thickness direction and reacts with a physiological environment to form a weak alkaline microenvironment, M metal materials exist in the form of simple substances of M, and M is calcium and/or strontium; the outer layer comprises an oxide of N which reacts with water to form a strong alkaline microenvironment, wherein N is at least one of magnesium and zinc; the intermediate layer is provided with an M metal material in the form of a simple substance of M and an N metal material in the form of an oxide of N, wherein M is calcium and/or strontium, and N is at least one of magnesium and zinc; the distribution of the titanium element is increased progressively from the surface of the middle layer to the surface of the inner layer along the thickness direction of the middle layer, the distribution of the oxygen element is increased progressively and then decreased progressively from the surface of the middle layer to the surface of the inner layer along the thickness direction of the middle layer, the distribution of the M element is increased progressively from the surface of the middle layer to the surface of the inner layer along the thickness direction of the middle layer, and the distribution of the N element is decreased progressively from the surface of the middle layer to the surface of the inner layer along the thickness direction of the middle layer; the composite coating on the surface of the titanium-based implant regulates the alkalinity size and the duration time of the microenvironment of the surface, so that the alkalinity of the microenvironment of the surface of the composite coating in a physiological environment is changed in a time sequence manner and a staged antibacterial osteogenesis effect is generated, wherein the degradation time of an oxide of N in the physiological environment is controlled by regulating the thickness of an outer layer of film, and the transition from strong to weak of the alkalinity microenvironment is brought by the element distribution of the middle layer.

2. The titanium-based implant surface composite coating as claimed in claim 1, wherein the outer layer has a thickness of 1-1000 nm.

3. The titanium-based implant surface composite coating as claimed in claim 1, wherein the M atomic percent of the inner layer is less than 20%, and the thickness of the inner layer is 1-150 nm.

4. The titanium-based implant surface composite coating as claimed in claim 1, wherein the thickness of the intermediate layer is 1-150 nm.

5. The titanium-based implant surface composite coating of claim 1, wherein the titanium-based implant is a titanium metal material, a titanium metal material having a titanium oxide coating on a surface thereof, a titanium metal material having at least one of a micro-scale structure, a sub-micro-scale structure, or a nano-scale structure on a surface thereof.

6. The titanium-based implant surface composite coating of claim 5, wherein the titanium metal material is pure titanium or a titanium alloy.

7. A method of surface modifying a titanium-based implant, comprising forming the titanium-based implant surface composite coating of any one of claims 1 to 6 by:

8. The method of claim 7, wherein step (1) comprises: m simple substance or M-Ti alloy is used as a cathode, the temperature of a vacuum chamber for plasma immersion ion injection is set to be 10-100 ℃, and the background vacuum degree is 1 multiplied by 10 ^-3 ～6×10 ^-3 Pa, the injection voltage is-5 to-50 kV, the frequency is 5 to 20Hz, the pulse width is 100 to 1000 mus, and the injection time is 0.5 to 240 minutes.

9. The method of claim 7, wherein step (2) comprises: applying radio frequency magnetron sputtering on the target material, setting the temperature of a vacuum chamber for magnetron sputtering to be 10-100 ℃, and the background vacuum degree to be 1 multiplied by 10 ^-3 ～6×10 ^-3 Pa, radio frequency power of 100-600W, and working air pressure of 1 × 10 ^-1 20Pa and 30-240 minutes of sputtering time.

10. The method of claim 7, wherein the titanium-based implant substrate is formed by at least one of grinding, turning, etching, additive manufacturing techniques prior to disposing the composite coating.