CN113117145A

CN113117145A - Antibacterial coating for surface of implant and preparation method thereof

Info

Publication number: CN113117145A
Application number: CN202010030836.1A
Authority: CN
Inventors: 曹辉亮; 罗启明; 刘宣勇; 马小涵
Original assignee: Shanghai Institute of Ceramics of CAS
Current assignee: Shanghai Institute of Ceramics of CAS
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2021-07-16

Abstract

The invention discloses an antibacterial coating for the surface of an implant and a preparation method thereof. The antibacterial coating is a composite nanorod array with a core-shell structure and vertically grown on the surface of the implant, the composite nanorod array comprises a zinc oxide nanorod and a shell layer formed on the zinc oxide nanorod in situ, and the shell layer is at least one of zinc sulfide, titanium oxide, iron oxide and ferrous sulfide; preferably, the shell layer is zinc sulfide.

Description

Antibacterial coating for surface of implant and preparation method thereof

Technical Field

The invention relates to an antibacterial coating for the surface of an implant, a preparation method and application thereof, in particular to an implant surface coating capable of controlling the release of zinc and a preparation method thereof, belonging to the field of surface modification of metal materials.

Background

The implant has two requirements on materials, namely, the implant has enough antibacterial property to maintain the tissues around the implant not to be infected due to the growth of bacterial biofilm so as not to cause the failure of the operation; secondly, the soft tissue sealing capability is required to be good, namely when a wound appears, cells at other parts can directionally move to the wound and adhere to and proliferate, and the sealing performance of a local area is ensured. Therefore, many researchers have been working on improving the antibacterial ability of dental implants, especially the antibacterial ability of their surfaces.

The antibacterial metal oxide nano-particles capable of being fixed on the surface of the matrix, such as a zinc oxide nano-rod array, is a strategy which can prevent the falling off, avoid the toxicity caused by the endocytosis of free nano-particles by cells and achieve the long-acting antibacterial effect. This way the antibacterial ability of the implant is a good way. However, the zinc oxide nanorod array has strong antibacterial effect and high toxicity to mammalian cells, and is not very good for cell adhesion [ patent US 2016/0175243 Al ] [ Biomaterials 2008,29: 3743-3749 ], so that it can be applied to the surface of an implant as an antibacterial coating, and the antibacterial property can be maintained at a high level while the cytotoxicity is reduced, so that it can be applied to the surface of the implant.

The toxicity of the zinc oxide nanorod array mainly comes from the massive release of zinc ions and the induction of apoptosis of cells by active oxygen substances generated on the surface of zinc oxide. Covering the surface of the zinc oxide nanorod array with a passivation layer shell can play a certain role in protecting a zinc oxide core and delay the release of zinc ions, but the shape of the nanorod array is easily damaged in this way [ Chinese patent CN 102793948A ], and the coating in the invention fills the gaps of the whole nanorod array structure, even if a few tips emerge, most of the rods are covered by calcium phosphate, so that the beneficial effect possibly brought by the shape of the rods is lost, and the adhesion and migration promotion effect of the shape on cells is further lost.

Disclosure of Invention

Aiming at the defects and the actual requirements of the implant in the prior art, the invention provides an antibacterial coating for the surface of the implant and a preparation method thereof, which overcome the problems of poor adhesion, short effective time or excessive cytotoxicity and the like of the traditional antibacterial coating.

In a first aspect, the invention provides an antibacterial coating for an implant surface, the antibacterial coating is a composite nanorod array with a core-shell structure and vertically grown on the implant surface, the composite nanorod array comprises a zinc oxide nanorod and a shell layer formed on the zinc oxide nanorod in situ, and the shell layer is at least one of zinc sulfide, titanium oxide, iron oxide and ferrous sulfide; preferably, the shell layer is zinc sulfide.

According to the invention, the shell layer grows in situ on the surface of the zinc oxide nanorod, the release rate of zinc oxide is further adjusted, the antibacterial property is ensured, the cytotoxicity is reduced, the original appearance of the coating is kept, and the soft tissue sealing capability of the material is promoted. Meanwhile, the nano rod-shaped array structure is beneficial to the adhesion and migration of cells, can provide more active sites and has better protein adhesion effect, which is beneficial to soft tissue sealing and can be better applied as a functional surface of a dental implant.

Preferably, the material of the implant comprises titanium, titanium alloy, nickel titanium alloy, stainless steel, tantalum, preferably titanium.

Preferably, the diameter of the composite nanorod with the core-shell structure is 50-160nm, and preferably 60-140 nm.

Preferably, the thickness of the antibacterial coating is 0.5-2 μm.

Preferably, spacing gaps exist among all the nanorods of the nanorod array; preferably, the shell layer completely covers the zinc oxide nanorod.

In a second aspect, the present invention also provides a method for preparing the antibacterial coating for the surface of the implant, the method comprising:

coating zinc oxide seed liquid on the surface of the implant and sintering at 400-600 ℃ to form a zinc oxide seed layer on the surface of the implant;

placing the implant with the zinc oxide seed layer in zinc oxide precursor solution, and growing a zinc oxide nanorod array on the surface of the implant by using a hydrothermal method;

and placing the implant with the zinc oxide nanorod array growing on the surface in a shell precursor solution corresponding to the shell, and reacting in a chemical bath at 40-80 ℃ for 3-12 hours to form a shell on the surface of each zinc oxide nanorod of the zinc oxide nanorod array.

Preferably, the zinc oxide seed solution is an alcoholic solution consisting of 0.02-0.08M ethanolamine and 0.05-0.1M zinc acetate dihydrate.

Preferably, the sintering heat preservation time is 20-40 minutes, and the heating rate is 5-10 ℃/min.

Preferably, the zinc oxide precursor solution is an aqueous solution consisting of 0.02-0.05M of hexamethylenetetramine and 0.02-0.05M of zinc nitrate hexahydrate; preferably, the hydrothermal temperature is 85-95 ℃, and the hydrothermal time is 2-6 h; more preferably, the method of inverted hydrothermal method is adopted, and the filling degree of the reaction kettle is 40-80%.

Preferably, the shell layer precursor solution is 0.02-0.05M thioacetamide aqueous solution, and the chemical bath temperature is 50-70 ℃.

Drawings

FIG. 1 is a scanning electron micrograph of a sample obtained by the treatment of examples 1 to 5, wherein (a), (b), (c), (d) and (e) in FIG. 1 correspond to examples 1, 2, 3, 4 and 5 respectively;

fig. 2 is an XRD spectrum of the sample treated in examples 1, 2, 3, 4 and 5, and (b) in fig. 2 is an enlarged view of (a) marked region in fig. 2;

FIG. 3 is a graph showing the release profile of zinc element from samples of examples 2, 3, 4, and 5 soaked in physiological saline;

FIG. 4 shows the results of the measurement of the amount of protein adsorbed on the surface of the samples of examples 1, 2, 3, 4 and 5;

FIG. 5 (a) is a graph of the plate-coating experiment of examples 1, 2, 3, 4, 5 on Staphylococcus aureus and Escherichia coli, FIG. 5 (b) is the inhibitory rate of Staphylococcus aureus in the above experiment, and FIG. 5 (c) is the inhibitory rate of Escherichia coli in the above experiment;

FIG. 6 shows the proliferation of human gingival fibroblasts after 1, 3 and 5 days of culture with samples treated in examples 1, 2, 3, 4 and 5;

FIG. 7 is a scanning electron micrograph of human gingival fibroblasts showing cell adhesion after 1, 3 and 5 days of culture to the samples treated in examples 1, 2, 3, 4 and 5;

FIG. 8 is the number of human gingival fibroblasts migrating per square millimeter after 6 and 12 hours of scratching in the scratched area on the samples treated in examples 1, 2, 3, 4, and 5;

FIG. 9 is a TEM image of the composite nanorods of example 4, wherein (a) in FIG. 9 is a morphology observation and a linear distribution of elements of the composite nanorods, and (b) in FIG. 9 is a high resolution transmission electron microscopy image and an electron diffraction analysis of a selected region of a white frame in (a).

Detailed Description

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

The invention prepares an antibacterial coating consisting of zinc oxide and a passivation layer shell on the surface of a dental implant. Specifically, the antibacterial coating is a core-shell structure composite nanorod array vertically growing on the surface of a plant body. The coating has good antibacterial effect and good biocompatibility, and is beneficial to adhesion and migration of human gingival fibroblast.

The core-shell structure composite nanorod array is obtained by growing in situ on the surface of a zinc oxide nanorod to form a shell. The in-situ growth can furthest reserve the appearance of the original zinc oxide nanorod array and reserve the promotion effect of the appearance on cell adhesion and migration. The shell is an oxide and/or sulfide shell. In some examples, the outer shell is at least one of zinc sulfide, titanium oxide, iron oxide, ferrous sulfide. The different components of the coating exist in a rod-shaped core-shell structure. In addition, the shell is formed by discontinuous nano-crystalline grain stacking, and the size of the crystalline grain is 1-10 nm. The shell layer is a continuous shell layer formed by discontinuous nanocrystalline accumulation. Discontinuous nanocrystals refer to single crystals or large crystal masses that are not grown continuously into zinc sulfide, and are not continuous amorphous, but rather a shell (shown in fig. 9) built up from one grain with a size of a few nanometers.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for regulating and controlling the antibacterial property and cytotoxicity of a zinc oxide nanorod array growing on the surface of an implant by using a passivation layer shell. The antibacterial coating formed by the preparation method has good antibacterial property and lower cytotoxicity, and the appearance of the nano rod-shaped structure improves the adhesion and migration of cells. The invention prepares the coating by means of coating sintering, hydrothermal and chemical bath.

The passivation layer is preferably zinc sulfide. The zinc sulfide is simple to prepare, and the precipitation equilibrium constant of the zinc sulfide in a neutral aqueous solution is far lower than that of the zinc oxide, so that the zinc sulfide represents lower dissolution amount, and can effectively serve as a passivation layer to play a role in slowing the dissolution speed of the zinc oxide.

Firstly, taking an implant material as a substrate, and preparing a zinc oxide seed layer on the surface of the implant by a coating and sintering mode. Such coating means include, but are not limited to, spin coating, and the like. The seed solution for coating is an alcohol solution consisting of 0.02-0.08M ethanolamine and 0.05-0.1M zinc acetate dihydrate. In some examples, the spin coating process is: the spin coating time is 10-30s, the rotation speed is 3000-6000rpm/min, the spin coating times are 1-5 times, and the single dropping amount is 30-90 mu L. Moreover, the sintering temperature can be 400-600 ℃, the heat preservation time is 20-40 minutes, and the heating rate is 5-10 ℃/min. And cooling along with the furnace after sintering.

And then, growing a layer of zinc oxide nanorod array on the surface of the implant attached with the zinc oxide seed layer by a hydrothermal method, and endowing the implant with antibacterial property. The hydrothermal solution is an aqueous solution consisting of 0.02-0.05M hexamethylenetetramine and 0.02-0.05M zinc nitrate hexahydrate. The hydrothermal temperature can be 85-95 ℃, and the hydrothermal time can be 2-6 h. In the specific implementation mode, an inverted hydrothermal method is adopted, and the filling degree of a reaction kettle is 40-80%. Inversion of hydrothermal method can avoid nucleation of zinc oxide in solution on surface to inhibit growth of zinc oxide nano rod.

And finally, converting the zinc oxide nanorod array into a core-shell structure nanorod array consisting of zinc oxide and a passivation layer shell by using a chemical bath method, and reducing cytotoxicity brought by the zinc oxide while keeping a rod-shaped structure. In some examples, the chemical bath solution is 0.02-0.05M thioacetamide aqueous solution, the chemical bath time can be 3-12h, preferably 6-12 h, and the chemical bath temperature can be 50-70 ℃. The chemical bath reaction can be carried out in a shaker, the shaker providing a rotation speed of 50-150rpm for the chemical bath.

In some embodiments, the implant may be pre-treated prior to preparing the zinc oxide seed layer on the surface of the implant. For example, it is cleaned by ultrasonic cleaning with an organic reagent and pure water and dried.

The composite coating in the Chinese patent CN 102793948A does not have a nano rod array structure, and is a coating with an irregular structure added on the basis of a rod array, wherein except some exposed zinc oxide tips, the other rod bodies are buried by calcium phosphate. The nano rod array structure is formed by self reaction instead of an additional coating, the original zinc oxide nano rod array structure is basically and completely stored, the rods are obviously separated except for the tips, the condition that the rods are connected does not exist, and the intervals among the rods are also stored. Moreover, as seen from fig. 1 in the chinese patent CN 102793948A, its zinc oxide is not completely covered, and its structure does not have the characteristics of the nano rod array structure.

Further, chinese patent CN 102793948A is a calcium phosphate coating prepared by spin-coating an exogenous precursor and sintering, and the present invention is a zinc sulfide shell generated by an exchange reaction between sulfur ions in an aqueous solution and oxygen in zinc oxide, which is a formation mechanism using zinc oxide as one of reaction sources.

The preparation method of the Chinese patent CN 110142049A is only suitable for free nanorods, and the method continues to provide an additional sulfur source and a zinc source after the cadmium sulfide is prepared, so that high-strength stirring is required to ensure uniformity, and the stirring is not suitable for uniformly modifying a surface coating and is more suitable for free particles; the invention provides a zinc source from the material, so the stirring step is avoided, and the method is suitable for the nanorod array growing on the surface. In addition, the invention aims at the modification of the zinc oxide nano rod, and the zinc oxide nano rod core which plays the main antibacterial effect has the zinc controlled release capacity.

The implant antibacterial coating prepared by the invention has a layer of nano-scale rod-shaped array, the diameter of the nano-scale rod-shaped array is 50-160nm, the length of the nano-scale rod-shaped array is 0.5-2 mu m, and rod-shaped structures are basically vertical to the surface of the implant, are uniformly distributed and are rarely adhered with each other. The cross section of the rod is regular hexagon or round. The coating has excellent antibacterial effect, obviously reduced cytotoxicity compared with pure zinc oxide, and is more favorable for cell adhesion and migration. In some examples, the invention improves cell adhesion and migration by keeping the nanorod structures and keeping the cumulative zinc release amount less than or equal to 0.2ppm in one day and less than or equal to 0.4ppm in one week (10 mL of physiological saline environment per square centimeter).

The implant antibacterial coating prepared by the invention has a layer of nano-scale rod-shaped array, the diameter of the nano-scale rod-shaped array is 50-160nm, the length of the nano-scale rod-shaped array is 0.5-2 mu m, and rod-shaped structures are basically vertical to the surface of the implant, are uniformly distributed and are rarely adhered with each other. The cross section of the rod is regular hexagon or round. The coating has excellent antibacterial effect, obviously reduced cytotoxicity compared with pure zinc oxide, and is more favorable for cell adhesion and migration.

Example 1

Polishing a medical grade titanium sheet with the size of 10mm in length, 10mm in width and 1mm in height by using abrasive paper, sequentially ultrasonically cleaning the medical grade titanium sheet by using acetone, absolute ethyl alcohol and ultrapure water for 15min each time, and then drying the medical grade titanium sheet in a 60 ℃ drying oven. The label is Ti.

FIG. 1a is the surface topography of the sample of example 1. Scanning electron microscope pictures, the visible surface is smooth and flat, and no obvious scratch exists. Fig. 2 shows the XRD pattern of example 1, consistent with pure titanium metal.

Example 2

The seed solution for the sample obtained in example 1 (an alcohol solution containing 50mM ethanolamine and 50mM zinc acetate dihydrate) was spin-coated at 4000rpm/min for 15 seconds in a dropping amount of 60. mu.L for 3 times. Placing the mixture into a tube furnace, sintering for half an hour at 500 ℃, heating at a rate of 5 ℃/min, and cooling along with the furnace after the temperature is increased. The sample was then poured into an aqueous hot solution (aqueous solution containing 20mM zinc nitrate hexahydrate and 20mM hexamethylenetetramine), reacted at 90 ℃ for 5 hours and then washed with ultrapure water to obtain a sample, labeled Ti-ZnO.

FIG. 1b is a scanning electron micrograph of the surface topography of the sample of example 2 showing that the surface has uniformly distributed zinc oxide nanorods, the size of which is about 64.6 (+ -16.6) nm, and the surface has a hexagonal prism shape, and the XRD pattern of Ti-ZnO is shown in FIG. 2. it can be seen that zinc oxide has a peak representing the (002) plane at 34.4 degrees, indicating that the growth mode of zinc oxide is oriented along the c-axis.

Example 3

The sample of example 2 was placed in a chemical bath solution (containing 40mM thioacetamide in water) and reacted in a shaker at 60 ℃ for 3 hours at 50 rpm. The resulting sample was labeled Ti-ZnO @ ZnS-3.

FIG. 1c is a scanning electron micrograph of the surface topography of the sample of example 3. The rod-like surface was seen to be roughened, increasing in diameter to about 94.1 (+ -12.6) nm. An XRD (X-ray diffraction) spectrum of Ti-ZnO @ ZnS-3 is shown in figure 2, and the characteristic peak intensity of a zinc oxide (002) surface is reduced, and an amorphous zinc sulfide package appears near 27 degrees. Example 3 still maintained the rod array structure.

Example 4

The sample of example 2 was placed in a chemical bath solution (containing 40mM thioacetamide in water) and reacted for 6 hours in a shaker at 60 ℃ with a shaker speed of 50 rpm. The resulting sample was labeled Ti-ZnO @ ZnS-6.

FIG. 1d is a SEM image of the surface topography of the sample of example 4. The rod diameter was further increased, approximately 125.7 (+ -13.8) nm, compared to Ti-ZnO @ ZnS-3, indicating a further widening of the zinc sulfide shell. The XRD pattern of Ti-ZnO @ ZnS-6 is shown in FIG. 2, and it can be seen that the characteristic peak intensity of the (002) face of zinc oxide is further reduced compared with the sample of example 3.

Example 5

The sample of example 2 was placed in a chemical bath solution (containing 40mM thioacetamide in water) and reacted for 9 hours in a shaker at 60 ℃ with a shaker speed of 50 rpm. The resulting sample was labeled Ti-ZnO @ ZnS-9.

FIG. 1e is a SEM image of the surface topography of the sample of example 5. The diameter of the rod continues to increase, approximately 133.1 (+ -16.0) nm, compared to Ti-ZnO @ ZnS-6, indicating a further widening of the zinc sulfide shell. The XRD pattern of Ti-ZnO @ ZnS-9 is shown in FIG. 2, and it can be seen that the characteristic peak of the (002) face of zinc oxide has disappeared completely, indicating that zinc oxide has disappeared completely and converted into zinc sulfide.

Example 6 Zinc content Release test

Examples 2, 3, 4 and 5 were soaked in physiological saline for 0.5, 1, 3, 5 and 7 days, and the solution was changed again at each time point, and the content of zinc element was measured by an inductively coupled plasma spectrometer.

Fig. 3 shows the cumulative release of zinc element as a function of time. It can be seen that the release rate of the zinc element of the Ti-ZnO is the fastest, and the platform period does not appear until 7 days, the release rates of the Ti-ZnO @ ZnS-3, the Ti-ZnO @ ZnS-6 and the Ti-ZnO @ ZnS-9 are reduced in sequence, and the release rates of the Ti-ZnO @ ZnS-3, the Ti-ZnO @ ZnS-6 and the Ti-ZnO @ ZnS-9 are reduced within three days. The difference among the groups is obvious, and the release rate of the zinc content is regulated and controlled.

Example 7 protein adsorption assay

The samples of examples 1, 2, 3, 4 and 5 were immersed in 2mg/L fetal bovine serum albumin solution, placed in an oven at 37 ℃, washed after 24 hours, and then eluted with protein, and the absorbance was measured to calculate the amount of protein adsorbed by the samples.

FIG. 4 shows the protein adsorption contents in examples 1, 2, 3, 4 and 5, and it can be seen that the proteins of Ti-ZnO, Ti-ZnO @ ZnS-3, Ti-ZnO @ ZnS-6 and Ti-ZnO @ ZnS-9 having rod-like structures have higher adsorption amounts than Ti, and the latter three are higher than Ti-ZnO, and there is no significant difference therebetween.

Example 8 antibacterial property test

Inoculating Staphylococcus aureus (or Escherichia coli) with good growth state into NB (or LB) liquid culture medium containing 25g/L nutrient broth culture medium (LB containing 10g/L tryptone and 5g/L yeast)Extract, 10g/L sodium chloride), the bacterial absorbance was measured using a microplate reader, whereby the bacterial concentration was formulated to 1X 10⁷CFU/mL for use. Respectively inoculating 60 mu L of bacterial suspension on the surfaces of Ti, Ti-ZnO @ ZnS-3, Ti-ZnO @ ZnS-6 and Ti-ZnO @ ZnS-9, culturing at 37 ℃ for 24h, shaking and falling staphylococcus aureus (or escherichia coli) from the surface of a sample, diluting bacterial liquid by 10 times, and inoculating 100 mu L of diluent on an NB (or LB) agar plate (the formula of the agar plate is that agar of 12g/L is additionally added corresponding to a liquid culture medium). After incubation at 37 ℃ for 16h, the number of bacteria in each group was counted, and the antibacterial ratio was calculated from the number of bacteria on the agar plate. The antibacterial rate is (number of colonies in control group-number of colonies in experimental group) ÷ number of colonies in control group × 100%, where Ti is the control group. The plating results and the antibacterial ratio between the groups are shown in fig. 5. It can be seen that Ti-ZnO, Ti-ZnO @ ZnS-3 and Ti-ZnO @ ZnS-6 have an antibacterial rate close to 100% against both bacteria.

Example 9 cell Activity assay

Evaluation of the Effect of the modified titanium materials obtained in examples 1, 2, 3, 4 and 5 on the cell viability Using Human Gingival Fibroblast (HGFs) in vitro culture experiments using Almarblue^TM) And detecting the proliferation of the cells on the surface of the material. The method comprises the following steps:

step A) the sterilized samples were placed in 24-well plates and 1mL of 5X 10 density medium was added dropwise to each well⁴cell/mL cell suspension, each group having at least three replicates;

step B) Place the cell culture plate in 5% CO₂Incubating in a cell culture box at 37 ℃ and saturated humidity for 24 h;

and C) after the cells are cultured for 1, 3 and 5 days, absorbing the original culture solution, adding a new culture solution containing 5% of Alma blue dye solution, placing the culture plate in an incubator for culturing for 2 hours, taking out 100 mu L of the culture solution from each hole, placing the culture solution into a 96-hole plate, and measuring 590nm fluorescence intensity of each hole under the excitation of 560nm by using an enzyme labeling instrument, wherein the fluorescence intensity is in direct proportion to the activity of the cells. FIG. 6 is a test result, and it can be seen that the cell activities of Ti-ZnO @ ZnS-3, Ti-ZnO @ ZnS-6, and Ti-ZnO @ ZnS-9 were sequentially increased compared to zinc oxide nanorods, indicating that the cytotoxicity of the material was sequentially decreased.

Example 10 cell adhesion

The influence of the titanium material obtained by the modification treatment of the above examples 1, 2, 3, 4 and 5 on cell adhesion is evaluated by adopting an in vitro culture experiment of Human Gingival Fibroblasts (HGFs), and the proliferation condition of the cells on the surface of the material is observed by using an electron scanning microscope. The method comprises the following steps:

step C) after 1, 3 and 5 days of cell culture, the original culture solution was aspirated, 4% glutaraldehyde solution was added and fixed overnight in a dark environment. And then dehydrating and drying the mixture by using 30% alcohol, 50% alcohol, 75% alcohol, 90% alcohol, 95% alcohol, 100% alcohol and a 2:1 alcohol/hexamethylene silane mixed solution, a 1:2 alcohol/hexamethylene silane mixed solution and pure hexamethylene silane, and observing the appearance and the adhesion condition on the surface of the sample by using an electron scanning microscope. The results in FIG. 7 show that after one day of incubation, the cells on Ti-ZnO @ ZnS-6 and Ti-ZnO @ ZnS-9 were significantly more numerous than the samples on Ti and spread well, indicating that the coating was favorable for cell adhesion. Cell coverage area ratios were 19.88 (+ -3.09%), 0.70 (+ -0.16%), 12.04 (+ -1.57%), 27.64 (+ -4.98%), and 28.95 (+ -1.96%) per day, respectively.

Example 11 cell migration

The influence of the titanium material obtained by the modification treatment of the above examples 1, 2, 3, 4 and 5 on cell migration was evaluated by an in vitro culture experiment of Human Gingival Fibroblasts (HGFs), and the cell migration ability was evaluated by a wound healing experimental method and observed by a fluorescence microscope. The method comprises the following steps:

step A) the sterilized samples were placed in 24-well plates and 1mL of 2X 10 density medium was added dropwise to each well⁵cell/mL cell suspension;

and B) after culturing for 40h, drawing a fine line on the surface of the sample on which the biofilm is formed by using a gun head, and continuing culturing after liquid is changed.

And C) continuing to culture for 6 or 12 hours, adding a 4% glutaraldehyde solution, fixing for 10min, staining with rhodamine-phalloidin and 4', 6-diamidino-2-phenylindole (DAPI), and observing and counting the cells in the scratched area by using a fluorescence microscope.

The results are shown in FIG. 8, where Ti-ZnO @ ZnS-6 has similar migration ability compared to Ti, while Ti-ZnO @ ZnS-9 has significantly better cell migration ability than Ti, indicating that the coating is favorable for cell migration. The number of migrated cells per square mm after 12 hours of streaking was 453.26 (+ -49.51), 9.83 (+ -4.73), 211.97 (+ -21.89), 598.93 (+ -111.52), 684.17 (+ -40.96), respectively.

Claims

1. The antibacterial coating for the surface of the implant is characterized in that the antibacterial coating is a composite nanorod array which vertically grows on the surface of the implant and has a core-shell structure, the composite nanorod array comprises a zinc oxide nanorod and a shell layer formed on the zinc oxide nanorod in situ, and the shell layer is at least one of zinc sulfide, titanium oxide, iron oxide and ferrous sulfide; preferably, the shell layer is zinc sulfide.

2. Antimicrobial coating according to claim 1, characterized in that the material of the implant comprises titanium, titanium alloy, nitinol, stainless steel, tantalum, preferably titanium.

3. The antibacterial coating according to claim 1 or 2, wherein the diameter of the composite nanorods with core-shell structure is 50-160nm, preferably 60-140 nm.

4. Antimicrobial coating according to any of claims 1 to 3, characterized in that the thickness of the antimicrobial coating is 0.5-2 μm.

5. The antimicrobial coating of any one of claims 1 to 4, wherein there are spacing voids between individual nanorods of the nanorod array; preferably, the shell layer completely covers the zinc oxide nanorod.

6. A method for preparing an antibacterial coating for an implant surface according to any one of claims 1 to 5, characterized in that it comprises:

7. The method according to claim 6, wherein the zinc oxide seed solution is an alcohol solution consisting of 0.02 to 0.08M ethanolamine and 0.05 to 0.1M zinc acetate dihydrate.

8. The method according to claim 6 or 7, wherein the sintering is carried out at a holding time of 20 to 40 minutes and a heating rate of 5 to 10 ℃/min.

9. The production method according to any one of claims 6 to 8, wherein the zinc oxide precursor solution is an aqueous solution composed of 0.02 to 0.05M of hexamethylenetetramine and 0.02 to 0.05M of zinc nitrate hexahydrate; preferably, the hydrothermal temperature is 85-95 ℃, and the hydrothermal time is 2-6 h; more preferably, the method of inverted hydrothermal method is adopted, and the filling degree of the reaction kettle is 40-80%.

10. The method according to any one of claims 6 to 9, wherein the shell layer precursor solution is a 0.02-0.05M aqueous thioacetamide solution, and the chemical bath temperature is 50-70 ℃.