CN114870083B - Preparation method and application of implant with complex coating on surface - Google Patents
Preparation method and application of implant with complex coating on surface Download PDFInfo
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- CN114870083B CN114870083B CN202210400588.4A CN202210400588A CN114870083B CN 114870083 B CN114870083 B CN 114870083B CN 202210400588 A CN202210400588 A CN 202210400588A CN 114870083 B CN114870083 B CN 114870083B
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- complex coating
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/06—Titanium or titanium alloys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/30—Inorganic materials
- A61L27/306—Other specific inorganic materials not covered by A61L27/303 - A61L27/32
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Landscapes
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
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- Dermatology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Transplantation (AREA)
- Inorganic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Composite Materials (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention provides an implant with a complex coating on the surface, wherein a phytic acid-metal ion complex coating is attached to the surface of the implant. The coating is constructed on the surface of the implant by combining metal-organic phosphate coordination bonds and interfacial restraint effect induced by solvent evaporation. The invention further provides a preparation method of the implant with the complex coating on the surface and application of the implant with the complex coating on the surface as an oral bone implant. The preparation method and the application of the implant with the complex coating on the surface are simple, efficient and environment-friendly preparation modes of the implant surface coating, the coating constructed by natural organic molecules and biological functional metal ions not only has good hydrophilicity and biocompatibility, but also has the multiple effects of resisting bacteria, depositing hydroxyapatite in situ, promoting osteogenesis and the like on the modified implant surface, and the preparation method is expected to become a method with development prospect for improving the implant implantation success rate.
Description
Technical Field
The invention belongs to the technical field of biological materials, relates to a preparation method and application of an implant with a complex coating on the surface, and in particular relates to a preparation method and application of an implant with a multifunctional phytic acid-metal ion complex coating material with antibacterial and bone promoting effects on the surface.
Background
The antibacterial property and the surface-interface osteogenesis activity are core requirements of an ideal bone implant material, however, the oral implant is mainly made of pure titanium, the material itself does not have the antibacterial property, and the deposition of hydroxyapatite (HAp) is hardly induced, and the sufficient osseointegration between the implant and the bone tissue is difficult to achieve (Yang, b.c., methou, x.d., yu, h.y., et al, advanced in titanium dental implant surface modification, huaxi kouqixue zazhhi.2019, 37 (2): 124-129.). Therefore, modifying the surface of the implant, by reducing bacterial adhesion and formation of a biological film, and promoting a good mineralization layer on the surface of the implant, enhancing osseointegration, will be beneficial to improving the success rate of the implant. The existing preparation process of the implant surface coating is generally complex, time-consuming and high in cost, and professional instruments and equipment are needed. Therefore, the provided preparation method of the implant surface coating has important clinical significance and is simple and practical to operate.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a simple, efficient and environment-friendly preparation method and application of an implant with a complex coating, so as to achieve the multifunctional effects of antibacterial and in-situ hydroxyapatite deposition and osteogenesis promotion on the surface of the modified implant, and solve the problems in the prior art.
To achieve the above and other related objects, a first aspect of the present invention provides an implant having a complex coating layer on a surface thereof, to which a phytic acid-metal ion complex coating layer is attached.
The second aspect of the present invention provides a method for preparing an implant having a complex coating on a surface thereof, comprising the steps of:
1) Rinsing and drying the implant after acid etching by adopting a first reagent to obtain an intermediate;
2) And repeating the procedures of dripping the complex working solution, drying, rinsing and drying at least once on the surface of the intermediate body to form at least one layer of complex coating.
In a third aspect, the present invention provides an implant having a complex coating on a surface thereof, prepared by the above method.
In a fourth aspect, the present invention provides the use of an implant having a coating of a complex on the surface thereof as an oral bone implant.
As described above, the preparation method and application of the implant with the complex coating on the surface provided by the invention have the following beneficial effects through the preferable formula components, the preparation steps and the conditions:
(1) The invention provides a preparation method and application of an implant with a complex coating on the surface, wherein the implant such as titanium surface is constructed with a phytic acid-metal ion complex such as PA-Fe/Cu complex coating which has good hydrophilicity and releases Cu in a two-stage mode 2+ 。
(2) The phytic acid-metal ion complex such as PA-Fe/Cu complex coating has obvious antibacterial effect on common pathogenic bacteria such as Porphyromonas gingivalis (P.gingivalis) in the oral cavity.
(3) The phytic acid-metal ion complex such as PA-Fe/Cu complex coating has good biocompatibility, and can promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).
(4) The invention provides a preparation method and application of an implant with a complex coating on the surface, wherein the phytic acid-metal ion complex such as PA-Fe/Cu complex coating can induce in-situ deposition of hydroxyapatite crystals on the titanium surface.
(5) According to the preparation method and application of the implant with the complex coating on the surface, compared with a control group, the phytic acid-metal ion complex coating such as the PA-Fe/Cu complex coating can generate more new bone tissues around the implant, and better bone combining capacity is shown.
(6) The preparation method and application of the implant with the complex coating on the surface provided by the invention are simple, efficient and environment-friendly, and the preparation method is a simple, efficient and environment-friendly preparation method of the titanium surface coating, can realize the multifunctional effects of resisting bacteria, depositing hydroxyapatite in situ, promoting osteogenesis and the like on the surface of the modified implant, and is expected to become a method with development prospect for improving the success rate of the implant.
Drawings
FIG. 1 is a schematic diagram showing the structure of the PA-Fe/Cu complex coating formed on the surface of the implant according to examples 1-2 of the present invention.
Fig. 2 shows the sectional morphology and thickness of PA-Fe/Cu complex coating layer on the surface of titanium sheet as implant sample in test example 1 of the present invention, wherein fig. 2a is a control group titanium sheet, fig. 2b is a PA-Fe/Cu2.5 complex coating layer group titanium sheet, and fig. 2c is a PA-Fe/Cu5 complex coating layer group titanium sheet, using a scanning electron microscope.
FIG. 3 is a graph showing the hydrophilicity of the PA-Fe/Cu complex coated titanium sheet of test example 1 according to the present invention compared with that of the control titanium sheet.
FIG. 4 shows the Fe of the PA-Fe/Cu complex coating of test example 1 according to the present invention 3+ And Cu 2+ Ion release profiles 4a, 4b, wherein FIG. 4a is Fe 3+ Ion release curve, FIG. 4b is Cu 2+ Ion release profile.
FIG. 5 shows the CFU count results of in vitro anti-P.gingivalis biofilm experiments on the surface of the PA-Fe/Cu complex coating of titanium plate as a sample of the implant in test example 2 of the present invention FIGS. 5a and 5b, wherein FIG. 5a is a photograph of colonies on blood plates of different experimental groups and control groups, and FIG. 5b is a graph of CFU quantitative count results.
FIG. 6 shows hydroxyapatite crystals formed on the surface of the PA-Fe/Cu complex coating layer on the surface of the titanium sheet as a implant sample in the physiological fluid environment in test example 3 of the present invention, wherein FIG. 6a is a control group titanium sheet and FIG. 6b is a PA-Fe/Cu5 complex coating layer group titanium sheet.
FIG. 7 is a graph showing the results of in vitro biocompatibility and cell proliferation experiments of a PA-Fe/Cu complex coating layer on the surface of a titanium sheet as a sample of an implant in test example 4 according to the present invention.
FIG. 8 shows the results of the test of BMSCs osteogenic related gene expression on the surface of the control titanium plate and PA-Fe/Cu complex coating in test example 4 of the present invention, wherein FIG. 8a is the expression level of osteogenic related gene Runx2, FIG. 8b is the expression level of osteogenic related gene COL-1α, FIG. 8c is the expression level of osteogenic related gene OPN, and FIG. 8d is the expression level of osteogenic related gene OCN.
FIG. 9 shows the results of Micro-CT scan and three-dimensional bone reconstruction of the PA-Fe/Cu complex coating modified implant in rat body in test example 5 of the present invention, wherein FIG. 9a is a simulation image of Micro-CT three-dimensional bone reconstruction, FIG. 9b is a quantitative statistical result image of the osteogenic index (BV), FIG. 9c is a quantitative statistical result image of the osteogenic index (BV/TV), FIG. 9d is a quantitative statistical result image of the osteogenic index (Tb.Th), and FIG. 9e is a quantitative statistical result image of the osteogenic index (Conn.Dn).
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
The first aspect of the present invention provides an implant having a complex coating on a surface thereof, to which a phytic acid-metal ion complex coating is attached.
The implant is a conventionally used oral implant. In particular, the implant is a threaded titanium pin made of commercially pure titanium.
In the implant, the body material of the implant is titanium. The titanium is pure titanium.
In the above implant, in the phytic acid-metal ion complex coating, the phytic acid is a conventionally used phytic acid (PA, CAS number 83-86-3). The phytic acid is an organic ligand which is well combined with the titanium substrate.
In the implant described above, in the phytic acid-metal ion complex coating, the metal ion is selected from at least one of iron ion or copper ion.
In one embodiment, the metal ions are iron ions and copper ions.
Specifically, the iron ions are ferric ions (Fe 3+ ). The iron ions are used as an inorganic cross-linking agent for connecting PA molecules, and the iron ions and the inorganic cross-linking agent are assembled in a coordinated manner to form a PA-Fe coating 'framework'.
Specifically, the copper ion is a divalent copper ion (Cu 2+ ). The copper ions have the capability of being released into the environment, and endow the coating with corresponding antibacterial and osseointegration-promoting multifunctional effects.
The second aspect of the present invention provides a method for preparing an implant having a complex coating on a surface thereof, comprising the steps of:
1) Rinsing and drying the implant after acid etching by adopting a first reagent to obtain an intermediate;
2) And repeating the procedures of dripping the complex working solution, drying, rinsing and drying at least once on the surface of the intermediate body to form at least one layer of complex coating.
In the step 1), the implant is subjected to ultrasonic cleaning in advance.
The ultrasonic cleaning is carried out in an ultrasonic oscillator.
In one embodiment, the cleaning agent for ultrasonic cleaning is acetone, absolute ethyl alcohol and water in sequence.
In a further embodiment, the washing time of each of said washing reagents is 14-16min, preferably 15min.
In the step 1), the first reagent is a mixed solution of sulfuric acid and hydrogen peroxide.
In one embodiment, the volume ratio of sulfuric acid to hydrogen peroxide in the first reagent is 0.9-1.1:1, preferably 1:1.
The sulfuric acid is an aqueous solution of sulfuric acid in an amount of 0.5 to 1.5mol/L, preferably 1 mol/L. The hydrogen peroxide is an aqueous solution of hydrogen peroxide with a volume percentage concentration of 25-35%, preferably 30%.
In the step 1), the temperature of the acid etching is room temperature.
In the above step 1), the acid etching is performed for 1 to 3 hours, preferably 2 hours.
The acid etching is immersing the implant in the first reagent.
In the step 1), the reagent used for rinsing is water. The water is used in an amount of 450-550mL, preferably 500mL.
In the above step 1), the number of rinsing times is 2 to 4, preferably 3; the time for each rinsing is 4-6min, preferably 5min.
In the above step 1), the drying is naturally air-dried at room temperature.
In one embodiment, the natural air drying time is 11 to 13 hours, preferably 12 hours.
In the above step 1), the above-mentioned dried product is stored at room temperature.
In the step 2), the complex working solution is a mixed solution of a phytic acid storage solution and a metal ion storage solution.
In one embodiment, the molar ratio of phytic acid in the phytic acid storage solution to metal ions in the metal ion storage solution is 6-13:5-13, such as 6.075-12.15:5.9-11.8, preferably 6.075:5.9 or 12.15:11.8, most preferably 12.15:11.8.
In a further embodiment, the metal ions in the metal ion storage solution are iron ions and copper ions, and the molar ratio of the phytic acid to the iron ions to the copper ions is 6-13:3-8:2-5, specifically, 6.075-12.15:3.7-7.4:2.2-4.4, preferably 6.075:3.7:2.2 or 12.15:7.4:4.4, and most preferably 12.15:7.4:4.4.
In one embodiment, the phytic acid storage solution is an ethanol dilution containing phytic acid solution.
Specifically, the phytic acid solution is 65-75% of phytic acid aqueous solution by volume percent. The water solution of phytic acid with the volume percentage of 65-75 percent is that the volume ratio of phytic acid to water is 65-75:25-35.
Specifically, in the phytic acid storage solution, the volume ratio of the phytic acid solution to the ethanol is 1:3.5-4.5, and is preferably 1:4.
In one embodiment, the metal ion storage liquid is a metal salt storage liquid.
Specifically, the metal salt storage liquid is at least one selected from ferric salt storage liquid and cupric salt storage liquid.
Further, the metal salt storage solution is a mixed solution of an iron salt storage solution and a copper salt storage solution.
Further, the ferric salt in the ferric salt storage solution is FeCl 3 ·6H 2 Ethanol solution of O.
Specifically, the concentration of iron ions in the iron salt stock solution is 1-10mg/mL, preferably 2.5-5mg/mL.
Further, the copper salt in the copper salt storage solution is CuCl 2 ·2H 2 Ethanol solution of O.
Specifically, the concentration of copper ions in the copper salt stock solution is 1-10mg/mL, preferably 2.5-5mg/mL.
In the step 2), the preservation temperature of the complex working solution is not more than 4 ℃, preferably 4 ℃.
In the step 2), the drop amount of the complex working solution on the surface of the intermediate is 4-5 mu L/cm 2 Preferably 4.68. Mu.L/cm 2 . The complex working solution spreads and spreads the whole intermediate surface.
In the above step 2), the drying is naturally performed at room temperature.
In one embodiment, the natural drying time is 5 to 15 minutes, preferably 10 minutes.
In the step 2), the reagent used for rinsing is absolute ethyl alcohol.
In the above step 2), the rinsing time is 8 to 12 minutes, preferably 10 minutes.
In the above step 2), the temperature of the drying is 75 to 85 ℃, preferably 80 ℃. The drying is performed in a drying oven.
In the above step 2), the drying time is 5 to 7 hours, preferably 6 hours.
In the step 2), the number of times of repeating the process is not less than 1.
The room temperature is 20-30 ℃.
The water is deionized water.
The ethanol is absolute ethanol.
In a third aspect, the present invention provides an implant having a complex coating on a surface thereof, prepared by the above method.
The preparation mechanism of the implant with the complex coating on the surface is as follows: and constructing the PA-Fe/Cu complex coating on the surface of the titanium by combining metal-organophosphorus coordination bonds and interfacial constraint effect induced by solvent evaporation. In the present invention, PA is withGood binding of titanium substrate to organic ligand, fe 3+ Then as an inorganic cross-linking agent for connecting PA molecules, the two are assembled in a coordinated way to form a PA-Fe coating 'framework', and Cu is introduced into the coating 2+ . Due to PA and Fe 3+ Is greater than Cu in combination 2+ More stable, thus Cu in the coating 2+ The coating has the capability of being released into the environment, and can endow the coating with corresponding antibacterial and osseointegration promoting multifunctional effects.
In a fourth aspect, the present invention provides the use of an implant having a coating of a complex on the surface thereof as an oral bone implant.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Example 1
The titanium nails used as the implant are sequentially cleaned by adopting acetone, absolute ethyl alcohol and water in an ultrasonic vibration instrument, and the cleaning time of each cleaning reagent is 15min. The titanium nails were then immersed in a 1:1 volume ratio sulfuric acid and hydrogen peroxide mixed solution and acid etched at room temperature for 2 hours. Wherein, the sulfuric acid is 1mol/L sulfuric acid aqueous solution, and the hydrogen peroxide is 30 percent hydrogen peroxide aqueous solution by volume percent concentration. Titanium nails are threaded titanium nails made from commercially pure titanium and are useful as human oral bone implants.
The acid etched implant was rinsed 3 times in 500mL deionized water for 5 minutes each time. Then, the intermediate samples were obtained by natural air-drying for 12 hours at room temperature. Intermediate samples 1 are stored at room temperature and the coating is to be prepared.
Drop 4.68. Mu.L/cm on the surface of intermediate sample 1 2 The complex working solution of (2) is spread and paved on the surface of the whole intermediate sample 1, naturally dried for 10min at room temperature, and the intermediate sample is rinsed for 1 min by absolute ethyl alcohol to remove free mattersFe of (2) 3+ 、Cu 2+ . And then placing the mixture in a drying oven at 80 ℃ for drying for 6 hours, so as to form a complex coating on the surface of the intermediate sample 1, and obtaining the implant sample 1 with the complex coating.
The complex working solution is a mixed solution of a phytic acid storage solution, an iron salt storage solution and a copper salt storage solution. Wherein the phytic acid storage solution is obtained by diluting 70% by volume of phytic acid aqueous solution with absolute ethyl alcohol according to the volume ratio of 1:4. The ferric salt stock solution is prepared by mixing 250mg FeCl 3 ·6H 2 O was dissolved in 50mL of absolute ethanol to obtain an iron salt stock solution having an iron ion concentration of 5mg/mL. The copper salt stock solution is prepared by adding 250mg of CuCl 2 ·2H 2 O was dissolved in 50mL of absolute ethanol to obtain a copper salt stock solution having a copper ion concentration of 5mg/mL. Then, the complex working solution was prepared by mixing 20. Mu.L of phytic acid stock solution, 400. Mu.L of ferric salt stock solution, and 150. Mu.L of copper salt stock solution to obtain a molar ratio of phytic acid, ferric ions, and copper ions of 12.15:7.4:4.4. The complex working solution is stored at 4 ℃. The structural schematic diagram of the PA-Fe/Cu complex coating formed on the surface of the implant is shown in figure 1.
Example 2
Selecting a titanium sheet or a titanium nail as a sample required by a performance test experiment of the implant, wherein the titanium sheet is a round commercial pure titanium sheet with the diameter of 14.75mm and the thickness of 1mm, and can be used for in vitro experiments; the titanium nail is a threaded titanium nail manufactured by commercial pure titanium, has the diameter of 2mm and the length of 6.5mm, and can be used for in vivo experiments of rats.
And cleaning the titanium sheet or the titanium nail in an ultrasonic vibration instrument sequentially by adopting acetone, absolute ethyl alcohol and water, wherein the cleaning time of each cleaning reagent is 15min. And immersing the titanium sheet or the titanium nail in a mixed solution of sulfuric acid and hydrogen peroxide in a volume ratio of 1:1, and carrying out acid etching for 2 hours at room temperature. Wherein, the sulfuric acid is 1mol/L sulfuric acid aqueous solution, and the hydrogen peroxide is 30 percent hydrogen peroxide aqueous solution by volume percent concentration.
The titanium sheet or titanium nail after acid etching is rinsed 3 times in 500mL deionized water, and the rinsing time is 5min each time. Then, the intermediate samples were obtained by natural air-drying for 12 hours at room temperature. Intermediate samples 2 are stored at room temperature and the coating is to be prepared.
Drop 4.68 μl/cm on the surface of intermediate sample 2 2 The complex working solution of (2) is spread and paved on the surface of the whole intermediate sample 2, naturally dried for 10min at room temperature, and the intermediate sample is rinsed for 2min by absolute ethyl alcohol to remove free Fe 3+ 、Cu 2+ . And then drying the mixture in a drying oven at 80 ℃ for 6 hours, so as to form a complex coating on the surface of the intermediate sample 2, and obtaining the implant sample 2 with the complex coating.
The complex working solution is a mixed solution of a phytic acid storage solution, an iron salt storage solution and a copper salt storage solution. Wherein the phytic acid storage solution is obtained by diluting 70% by volume of phytic acid aqueous solution with absolute ethyl alcohol according to the volume ratio of 1:4. The ferric salt stock solution is prepared by mixing 250mg FeCl 3 ·6H 2 O was dissolved in 50mL of absolute ethanol to obtain an iron salt stock solution having an iron ion concentration of 5mg/mL. The copper salt stock solution is prepared by adding 250mg of CuCl 2 ·2H 2 O was dissolved in 50mL of absolute ethanol to obtain a copper salt stock solution having a copper ion concentration of 5mg/mL. Then, the complex working solution was prepared by mixing 20. Mu.L of phytic acid stock solution, 400. Mu.L of ferric salt stock solution, and 150. Mu.L of copper salt stock solution to obtain a molar ratio of phytic acid, ferric ions, and copper ions of 12.15:7.4:4.4. The complex working solution is stored at 4 ℃. The structural schematic diagram of the PA-Fe/Cu complex coating formed on the surface of the implant is shown in figure 1.
Example 3
A batch of 3 titanium sheets or nails was prepared using the preparation procedure of example 2 to obtain intermediate samples # 1, # 2, # 3.
The preparation procedure of example 2 was followed to obtain implant samples # 1 (labeled PA-Fe/Cu 1.25), # 2 (labeled PA-Fe/Cu 2.5) and # 3 (labeled PA-Fe/Cu 5) with a complex coating.
Wherein, the complex working solution 3# is the complex working solution selected in example 2. The molar concentration of the complex working solution 2# is 1/2 of the complex working solution 1# and the molar concentration of the complex working solution 1# is 1/4 of the complex working solution 3#.
The procedure of dropping the complex working solution, drying, rinsing and drying in example 2 was further performed 1 time on the surface of sample # 3 to prepare implant sample # 4 having a two-layer complex coating.
And the uncoated pure titanium sheet or titanium nail is used as a control group sample. Implant samples # 1, # 2, # 3, # 4 were used to screen coating materials with optimal antimicrobial and bone-promoting effects on the implant surface.
Test example 1
The thickness, hydrophilicity and ion release curve of the coating are detected by using a scanning electron microscope, a contact angle tester, an inductively coupled plasma atomic emission spectrum and the like.
The titanium sheet-based implant samples 2# and 3# prepared in example 3 and the control group samples were tested, the samples were embedded by a vacuum embedding machine and cut by an automatic grinding and polishing machine, the cross section of the titanium sheet was scanned by using a Scanning Electron Microscope (SEM), the cross section morphology of the PA-Fe/Cu complex coating was recorded by photographing, and the thickness of the coating was measured, and the specific results are shown in FIGS. 2a, 2b and 2c. As shown in FIGS. 2b and 2c, the coating thicknesses of the PA-Fe/Cu2.5 and PA-Fe/Cu5 samples were 246.67.+ -. 20.82nm and 643.33.+ -. 11.55nm, respectively.
In the hydrophilicity test, a water drop is dropped onto the surface of a material, and the hydrophilicity of the coating layer is determined by measuring the water contact angle by a contact angle meter. In the hydrophilicity test, the water contact angles of PA-Fe/Cu2.5, PA-Fe/Cu5 and the control group were 38.47.+ -. 0.81 °, 33.23.+ -. 1.31 °, and 135.52.+ -. 1.68 °, respectively, as shown in FIG. 3. Compared with the control group, the surface of the PA-Fe/Cu complex coating has better hydrophilicity.
Detection of Fe in PA-Fe/Cu complex coating by inductively coupled plasma atomic emission spectrometry (ICP-MS) 3 + 、Cu 2+ Release in Simulated Body Fluid (SBF). Briefly, the different groups of samples were immersed in 10mL of simulated body fluid, respectively, simulating an in vivo environment at 37 ℃,5% CO 2 SBF was collected at different time points sequentially after 1, 3, 7, 14, 21 days of incubation in the cell culture chambers, while fresh SBF was added to the centrifuge tube on days 1, 3, 7, 14 for continued incubation. As shown in FIG. 4a, fe 3+ The release amount on the first day was 5.31.+ -. 0.30ppb, and that on days 2-3 was 7.22.+ -. 0.51ppb, 5.70+ -0.52 ppb on days 4-7. As shown in FIG. 4b, the first day, cu 2+ The release amount of (2) was 23.64.+ -. 2.43ppb, the release amount was 14.21.+ -. 2.42ppb on days 2-3, and the release amount was 12.69.+ -. 1.88ppb on days 4-7. Cu (Cu) 2+ Exhibits a two-stage mode, exhibits a relatively high release over the first three days of soaking, releases relatively slowly over the next 2-3 weeks, and Fe 3+ The release behavior was relatively slow over a period of 3 weeks of soaking.
The results show that the PA-Fe/Cu complex coating structure can be successfully constructed on the surface of the titanium sheet serving as the simulated implant, and the constructed coating has good hydrophilicity and can release Cu in a two-stage mode 2+ 。
Test example 2
The number of viable P.gingivalis in the biofilm on the surface of the PA-Fe/Cu complex coating was evaluated by Colony Forming Unit (CFU) counting. 1mL was concentrated to 10 7 CFU/mL of the P.gingivalis suspension was inoculated into 24-well plates in which each group of titanium plates was placed, and cultured under anaerobic conditions at 37 ℃. Each group of titanium sheets was titanium sheet-based implant samples # 1, # 2, # 3, # 4 and control group samples prepared in example 3, respectively.
And (3) measuring the number of viable bacteria in the biological film on the surface of each coating after 24, 48 and 96 hours by adopting a CFU counting method, taking out corresponding 24 pore plates at different time points, discarding bacterial liquid in the pore plates, washing 3 times by using 1mL of Phosphate Buffer Solution (PBS), and removing bacteria which are not adhered to the surface of the titanium sheet. Taking out the titanium sheet in the hole under the sterile environment, placing the titanium sheet in a centrifuge tube, adding 1mL of sterile PBS into each tube, sealing, and performing ultrasonic vibration treatment to make bacteria adhered to the surface of the titanium sheet fall off. Bacterial liquid in the centrifuge tube is diluted by 10 times by PBS, evenly coated on blood agar plates for culturing for 4-5 days under the anaerobic condition of 37 ℃, and the number of colonies growing on each group of blood agar plates is quantitatively analyzed by counting, and one colony is recorded as one CFU.
The photographs of the colonies on the surface of the blood plates as shown in FIG. 5a show that the number of colonies on the PA-Fe/Cu complex coating layer is significantly smaller than that on the control group. As shown in FIG. 5b, at 24 hours, the viable count of the PA-Fe/Cu 1.25, PA-Fe/Cu2.5, PA-Fe/Cu5 and PA-Fe/Cu 5X 2 complex coating groups P.gingivalis was respectively reduced compared with the surface of the control titanium sheet1.37log less 10 ，1.88log 10 ，2.02log 10 And 2.68log 10 . At 48 hours, the number of the viable bacteria of the P.gingivalis on the surface of the coating of the experimental group is respectively reduced by 1.85log compared with that of the titanium sheet of the control group 10 ，3.92log 10 ，4.83log 10 And 6.37log 10 . At 72 hours, the number of P.gingivalis viable bacteria on the surface of each group of coatings was reduced by 0.38log, respectively 10 ，4.48log 10 ，6.39log 10 And 7.37log 10 。
The results show that the PA-Fe/Cu2.5, PA-Fe/Cu5 and PA-Fe/Cu5 multiplied by 2 complex coatings have obvious P.gingivalis biological film resisting effect.
Test example 3
Titanium flakes were immersed in SBF at ion concentrations close to human plasma and their in vitro osteogenic properties were evaluated by the ability of the material surface to form apatite. According to the literature, the volume (mL) of SBF is recommended as the sample surface area (mm 2 ) One tenth (Kokubo, T., takadama, H.How useful is SBF in predicting in vivo bone bioactivity biomaterials.2006,27 (15): 2907-2915.). The above titanium sheet was respectively a titanium sheet-based implant sample 3# prepared in example 3 and a control sample.
Briefly, each group of titanium sheets was immersed in 20mL SBF, with the coated titanium sheet facing downward. At 37℃and 5% CO 2 After taking all titanium plate samples from SBF, surface inorganic salts were removed by rinsing with a large amount of deionized water and dried at room temperature. The apatite formed on the surface of the different titanium sheet samples was examined using SEM. SEM examination showed that after soaking in SBF, as shown in fig. 6a, sporadic HAp crystal structure was seen on the surface of the control titanium sheet. Under the same experimental conditions, a layer of hemispherical HAp crystals with nanometer level is uniformly accumulated on the surface of the PA-Fe/Cu5 complex coating shown in FIG. 6 b.
The results show that the PA-Fe/Cu5 complex coating has the effect of promoting the in-situ deposition of HAp.
Test example 4
In vitro inoculation of mouse bone marrow mesenchymal stem cells (BMSCs), the biocompatibility of the coating on the BMSCs and the bone property are evaluated by methods such as cell proliferation, real-time fluorescence quantitative polymerase chain reaction (qPCR) and the like. Each group of titanium plates was titanium plate based implant samples # 2, # 3, # 4 and control samples.
BMSCs suspension concentration was adjusted to 5X 10 by microscopic counting 4 Each group of titanium plates was placed in 24-well plates, 1mL of cell suspension was added to each well, and cultured in a 37℃cell incubator for 1 day, 3 days, and 7 days, respectively. The CCK-8 kit was used to test the proliferation capacity of BMSCs cells adhered to the surface of the PA-Fe/Cu complex coating. The corresponding well plates were removed at various time points, the stock culture was aspirated, rinsed 3 times with PBS, 225. Mu.L of serum-free alpha-MEM and 25. Mu.L of CCK-8 solution were added to each well, and incubated at 37℃for 2 hours in the absence of light. Transferring the supernatant into a new 96-well plate, and detecting OD (optical density) by using an enzyme-labeled instrument 450nm Values. As shown in FIG. 7, on day 1 of the culture, the proliferation potency of BMSCs on the surface of the PA-Fe/Cu 5X 2 complex coating was lower than that of the control titanium plate on day 1, while the proliferation potency of BMSCs on the surfaces of the PA-Fe/Cu2.5 and PA-Fe/Cu5 complex coatings was not significantly different from that of the control titanium plate. On the 3 rd day of culture, the proliferation capacities of BMSCs on the surfaces of the PA-Fe/Cu2.5 and PA-Fe/Cu5 complex coatings are superior to those of the titanium plates of the control group, and the proliferation capacities of BMSCs on the surfaces of the PA-Fe/Cu5 complex coatings are also superior to those of the PA-Fe/Cu5 multiplied by 2 complex coatings, and the proliferation capacities of BMSCs cells on the surfaces of the PA-Fe/Cu5 multiplied by 2 complex coatings and the titanium plates of the control group are not obviously different. On day 7 of culture, the PA-Fe/Cu5 complex coating showed the best BMSCs proliferation promoting ability, while the BMSCs cell proliferation ability of the PA-Fe/Cu2.5 and PA-Fe/Cu 5X 2 complex coating surfaces were not significantly different from that of the control titanium plate.
The results show that the PA-Fe/Cu2.5 and PA-Fe/Cu5 complex coating has good biocompatibility and the effect of promoting the proliferation of BMSCs.
The effect of the PA-Fe/Cu complex coating on BMSCs osteogenesis-related gene expression was examined by qPCR. The concentration of BMSCs suspension was adjusted to 5X 10 4 Each group of titanium plates was placed in 24-well plates, 1mL of cell suspension was added to each well, and cultured in a cell incubator at 37℃for 7 days. According to manufacturer instructions, (1) cell collection; (2) extracting Total RNA; (3) reverse transcription of RNA; (4) and (5) quantitative PCR detection. qP by SYBR Green technologyCR assay was performed to amplify the expression of bone formation related gene mRNAs such as RUNX2, COL-1. Alpha., OPN and OCN, which are main markers of bone formation differentiation, respectively, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. And finally, ct value is read and statistical analysis is carried out. As shown in FIG. 8, the expression levels of Runx2 and OPN of BMSCs on the surface of the PA-Fe/Cu2.5 complex coating were significantly increased as compared with the control titanium sheet, wherein the expression level of Runx2 of BMSCs on the surface of the PA-Fe/Cu5 complex coating was the highest in the three groups. In addition, the expression level of COL-1 alpha and OCN of BMSCs on the surface of the PA-Fe/Cu5 complex coating is obviously increased, and the expression level of COL-1 alpha and OCN of BMSCs on the surface of the PA-Fe/Cu2.5 complex coating is not obviously different from that of a titanium sheet in a control group.
The results show that the PA-Fe/Cu2.5 and PA-Fe/Cu5 complex coatings have the potential to promote osteogenic differentiation of BMSCs.
Test example 5
And implanting a titanium nail with a PA-Fe/Cu complex coating into the femur of the rat, and constructing a femoral implantation model of the rat. The effect of bone union around the implant was analyzed 8 weeks after surgery by Micro-computed tomography (Micro-CT) and histological staining. Titanium staples were titanium staple based implant samples 2#, 3# and control samples prepared in example 3.
In vivo experiments in this study were approved by the ethics committee. Healthy male Sprague-Dawley rats of 10 weeks of age were randomly divided into three groups, namely a control group, a PA-Fe/Cu2.5 group and a PA-Fe/Cu5 group. The operation steps are as follows: (1) anesthesia: sulbactam, prochloraz and normal saline are mixed according to 1.6mL:0.4mL: mixing 8mL to prepare an anesthetic, and performing general anesthesia on rats in a mode of intraperitoneal anesthesia, wherein the dosage is 0.2mL/100g; (2) skin preparation: the hairs of the parts below the hip joint of the bilateral hind limbs of the rat are shaved. Disinfecting iodine tincture in the skin preparation area of the prone position of the rat, and spreading towel; (3) planting operation: cutting the skin at the outer side area of the knee joint, blunt separating muscle and periosteum, exposing the femoral head at the far end of the femur of the rat, preparing a planting nest with the depth of about 2mm by using a drill bit with the diameter of 1.8mm, placing the implant at an orifice, pushing the implant into the planting nest while rotating, and taking the flush bone surface at the junction of the crown and the thread of the implant as a standard for positioning; (4) stitching: after the implant is implanted and in place, the incision is tightly sutured in layers using 5-0 non-absorbable sutures. Day 3 post-surgery antibiotics were administered and rats were observed for wound status and systemic status.
Rats were sacrificed 8 weeks after surgery by intraperitoneal injection of 10% chloral hydrate, bone tissue pieces containing implants were removed, and 4% paraformaldehyde was fixed for 24 hours. Bone formation around the implant was analyzed by Micro-CT. The region of interest (region of interest, ROI) was set to 0.2mm around the implant. New bone formation was quantitatively analyzed by calculating Bone Volume (BV), volume/total volume (BV/TV), bone trabecular thickness (trabecular thickness, tb. Th) and bone trabecular junction density (connectivity density, conn. Dn). As shown in FIG. 9a, micro-CT scan results of bone tissue surrounding three groups of implants. From the three-dimensional images of the 4 different directions, it can be seen that the PA-Fe/Cu2.5 and PA-Fe/Cu5 complex coated implants produced more bone tissue around and between the threads than the control. Fig. 9b, 9c, 9d, 9e are indicators of quantitative analysis of bone tissue formation around each group of implants. The results showed that the Conn.Dn values of the PA-Fe/Cu2.5 complex coated implants were statistically different from the control group, and the BV, BV/TV, tb.Th and Conn.Dn values of the PA-Fe/Cu5 complex coated implants were statistically different from the control group.
The results show that the PA-Fe/Cu2.5 and PA-Fe/Cu5 complex coating implant has the effect of promoting surrounding osseointegration. However, compared with the implant coated with the PA-Fe/Cu5 complex, the implant has good hydrophilicity, antibacterial property and optimal performance, and promotes HAp in-situ deposition and osteogenesis.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (6)
1. A method for preparing an implant with a complex coating on the surface, comprising the following steps:
1) Rinsing and drying the implant after acid etching by adopting a first reagent to obtain an intermediate;
2) Repeatedly performing the procedures of dripping the complex working solution, drying, rinsing and drying at least once on the surface of the intermediate to form at least one layer of complex coating;
in the step 1), the first reagent is a mixed solution of sulfuric acid and hydrogen peroxide; in the first reagent, the volume ratio of the sulfuric acid to the hydrogen peroxide is 0.9-1.1:1;
in the step 1), the reagent adopted in the rinsing is water;
in the step 2), the complex working solution is a mixed solution of a phytic acid storage solution and a metal ion storage solution;
the phytic acid storage solution is ethanol diluent containing phytic acid solution; the phytic acid solution is 65-75% of phytic acid aqueous solution by volume percent; in the phytic acid storage solution, the volume ratio of the phytic acid solution to the ethanol is 1:3.5-4.5;
the metal ion storage liquid is a metal salt storage liquid; the metal salt storage solution is a mixed solution of an iron salt storage solution and a copper salt storage solution; the ferric salt stock solution is FeCl 3 ·6H 2 Ethanol solution of O; the copper salt storage solution is CuCl 2 ·2H 2 Ethanol solution of O;
the metal ions in the metal ion storage solution are iron ions and copper ions, and the mole ratio of the phytic acid to the iron ions to the copper ions is 6-13:3-8:2-5;
in the step 2), the dripping amount of the complex working solution on the surface of the intermediate is 4-5 mu L/cm 2 ；
In the step 2), the reagent adopted in the rinsing is absolute ethyl alcohol.
2. The method for preparing an implant with a complex coating on the surface according to claim 1, wherein in step 1), the implant is subjected to ultrasonic cleaning in advance; the cleaning reagent for ultrasonic cleaning is acetone, absolute ethyl alcohol and water in sequence.
3. The method for preparing an implant with a complex coating on the surface according to claim 1, wherein in the step 1), the conditions are as follows:
a1 The temperature of the acid etching is room temperature;
a2 The acid etching time is 1-3 hours;
a3 The rinsing times are 2-4 times;
a4 Naturally air-drying at room temperature, wherein the natural air-drying time is 11-13 hours;
a5 After said drying, preserving at room temperature.
4. The method for preparing an implant with a complex coating on the surface according to claim 1, wherein in the step 2), the conditions are as follows:
b1 Naturally drying at room temperature for 5-15min;
b2 The rinsing time is 8-12min;
b3 The temperature of the drying is 75-85 ℃;
b4 The drying time is 5-7 hours;
b5 The repeated times of the working procedures are more than or equal to 1 time.
5. An implant with a complex coating on the surface, wherein a phytic acid-metal ion complex coating is attached to the surface of the implant;
the body of the implant is made of titanium; the metal ions are iron ions and copper ions; the iron ions are ferric ions; the copper ions are bivalent copper ions;
the implant with the complex coating on the surface is prepared by the method of any one of claims 1-4.
6. Use of an implant according to claim 5 with a coating of a complex on the surface for the preparation of an oral bone implant.
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