阅读说明：本技术 一种医用植入物表面有机-无机抗菌复合涂层及其制备方法 (Organic-inorganic antibacterial composite coating on surface of medical implant and preparation method thereof ) 是由 桑婷 唐镇 李坤 王慧慧 于 2021-09-02 设计创作，主要内容包括：本发明涉及一种医用植入物表面有机-无机抗菌复合涂层及其制备方法,步骤如下：制备纳米银粉；在硼砂-氢氧化钠缓冲液中加入纳米银粉得到纳米银溶液,在水中加入抗菌肽GL13K得到GL13K溶液,接着将两者混合,在4℃下储存4天,GL13K自组装形成纳米纤维,得复合物溶液；将经过预处理的医用植入物放入48孔板中,每个孔板中加入250μL纳米银-抗菌肽GL13K复合物溶液,用封口膜封闭孔板边缘,在4℃下培养12 h后,从溶液中取出医用植入物,去离子水小心冲洗,氮气干燥后,即得复合涂层。本发明将纳米银和抗菌肽GL13K两者结合形成复合涂层,可达到优势互补的效果。(The invention relates to an organic-inorganic antibacterial composite coating on the surface of a medical implant and a preparation method thereof, and the preparation method comprises the following steps: preparing nano silver powder; adding nano silver powder into borax-sodium hydroxide buffer solution to obtain nano silver solution, adding antibacterial peptide GL13K into water to obtain GL13K solution, mixing the two solutions, storing at 4 ℃ for 4 days, and self-assembling GL13K to form nano fibers to obtain composite solution; putting the pretreated medical implant into 48-pore plates, adding 250 mu L of nano silver-antibacterial peptide GL13K compound solution into each pore plate, sealing the edge of each pore plate by using a sealing film, culturing at 4 ℃ for 12h, taking out the medical implant from the solution, carefully flushing with deionized water, and drying with nitrogen to obtain the composite coating. According to the invention, the nano-silver and the antibacterial peptide GL13K are combined to form the composite coating, so that the effect of complementary advantages can be achieved.)

1. A preparation method of an organic-inorganic antibacterial composite coating on the surface of a medical implant is characterized by comprising the following preparation steps:

(1) preparing nano silver powder: preparing nano silver powder by reducing silver nitrate by using citrate as a reducing agent and sugar as a stabilizing agent;

(2) preparation of nano silver-antibacterial peptide GL13K self-assembly nanofiber composite solution: adding nano silver powder into borax-sodium hydroxide buffer solution with the pH value of 9.8 to prepare 0.2mg/mL nano silver solution, adding antibacterial peptide GL13K into water, shaking uniformly to prepare 100mg/mL GL13K solution, then adding GL13K solution into the nano silver solution, wherein the volume ratio of GL13K solution to nano silver solution is 1:99, the final concentration of nano silver and GL13K in the obtained mixed solution is 0.2mg/mL and 1mg/mL respectively, storing the mixed solution at 4 ℃ for 4 days, namely, carrying out self-assembly of GL13K, and forming nano silver-antibacterial peptide GL13K self-assembled nanofiber composite solution;

(3) preparing an organic-inorganic antibacterial composite coating: putting the pretreated medical implant into 48-pore plates, adding 250 mu L of nano silver-antibacterial peptide GL13K compound solution into each pore plate, sealing the edge of each pore plate by using a sealing film, culturing at 4 ℃ for 12h, taking out the medical implant from the solution, carefully flushing with deionized water, and drying with nitrogen to form an organic-inorganic antibacterial composite coating on the surface of the medical implant.

2. The method for preparing the organic-inorganic antibacterial composite coating on the surface of the medical implant according to claim 1, wherein the specific preparation method of the nano silver powder in the step (1) is as follows: dropwise adding 250mM citric acid aqueous solution into 1mM silver nitrate aqueous solution, wherein the volume ratio of the citric acid solution to the silver nitrate solution is 1:50, stirring for 10min in a water bath at 93-94 ℃, centrifuging for 30min under the centrifugal force of 35280RCF, and repeatedly centrifuging for three times to collect precipitated nano-silver; then, dissolving the collected nano silver in deionized water again, centrifuging for 10min under the centrifugal force of 2380RCF, and collecting the precipitated nano silver by repeating the centrifugation for three times; diluting the finally collected nano-silver precipitate with deionized water, adding deionized water dropwise, and measuring Optical Density (OD) value with spectrophotometer until the precipitate is diluted to OD₄₂₀And =2.9, adding 0.1g of sucrose per mL, mixing and stirring for 2h, drying for 24h in a freeze dryer to obtain nano silver powder, sealing the nano silver powder, and storing at-20 ℃ for later use.

3. The preparation method of the organic-inorganic antibacterial composite coating on the surface of the medical implant according to claim 2, characterized in that: the citrate in the step (1) is trisodium citrate.

4. The preparation method of the organic-inorganic antibacterial composite coating on the surface of the medical implant according to claim 1, characterized in that: the borax-sodium hydroxide buffer solution in the step (2) is prepared from 0.025M borax water solution and 0.1M NaOH water solution.

5. The method for preparing the organic-inorganic antibacterial composite coating on the surface of the medical implant according to claim 1, wherein the pretreatment method of the medical implant in the step (3) is as follows: soaking the medical implant in acetone for 10min, ultrasonically cleaning with deionized water for 15min, ultrasonically treating with cyclohexane for 15min, and blow-drying with nitrogen; then the medical implant is put into a test tube, 5mL of NaOH aqueous solution with the concentration of 5M is added into the test tube, the test tube is covered and fixed, the test tube is placed in a 60 ℃ incubator for alkali etching for 12 hours, then the medical implant is washed by deionized water and acetone in sequence, and then dried by nitrogen for standby.

6. The preparation method of the organic-inorganic antibacterial composite coating on the surface of the medical implant according to claim 5, wherein the preparation method comprises the following steps: the medical implant is a Ti6Al4V micro-implant.

7. An organic-inorganic antibacterial composite coating on the surface of a medical implant prepared by the preparation method of any one of claims 1-6.

Technical Field

The invention relates to the technical field of medical materials, in particular to an organic-inorganic antibacterial composite coating on the surface of a medical implant and a preparation method thereof.

Background

Infection is one of the most serious complications of various metal implantation operations (titanium plates, titanium nails, implants, implantation nails, artificial joints and the like), often causes implant implantation failure, and is a problem to be solved urgently. The most critical pathogenic factor in the development of implant infection is the formation of bacterial biofilm, which begins immediately after bacterial adhesion and has very strong antibiotic resistance once the bacteria adhere and aggregate on the surface of the implant to form a bacterial biofilm. According to the theory of 'competitive surface', bacteria and host cells are planted on the surface of the implant in a competitive mode, if the implant material has antibacterial capacity, the competitive power of the bacteria can be weakened in the early stage of implantation, more planting time is strived for the host cells, and the success rate of implantation is greatly improved. The titanium surface antibacterial modified coating capable of reducing infection is a research hotspot at present, researchers try to add different antibacterial substances such as antibiotics (gentamicin, minocycline rifampicin and the like), non-antibiotic organic antibacterial agents (chlorhexidine, chloroxylenol), nanoparticles (silver, copper, zinc oxide) and the like into the coating to endow the implant with the capabilities of inhibiting bacterial adhesion and killing bacteria to prevent and control the related infection of the implant, although the coating method is various, the coating method still has the problems of poor antibacterial property, drug resistance, complex operation and the like, and a high-efficiency and reliable solution is needed urgently.

Antimicrobial peptides (AMPs) are natural organic polypeptides with immunological activity generated in a human body, and are considered to be natural new-generation antibiotics due to unique antibacterial mechanism, broad-spectrum antibacterial activity on drug-resistant strains and difficulty in generating new drug-resistant bacteria. Antibacterial peptide GL13K is a positive charge amphipathic small molecule polypeptide extracted from human parotid gland secretory protein, and in recent years, its antibacterial action on the surface of titanium implant has made a breakthrough research result, and its coating on the surface of titanium implant can inhibit the growth of biological membrane and kill several related pathogens. Most importantly, the antibacterial composition can effectively kill various drug-resistant bacteria without causing new drug-resistant bacteria. Meanwhile, the coating is firmly combined with the titanium implant (has the capability of resisting hydrolysis and mechanical challenge), has good cell compatibility (has no cytotoxicity to osteoblasts, human gingival fibroblasts, red blood cells and other cells), and has a simple and easy coating method.

However, antimicrobial peptides are passive organic antimicrobial coatings whose antimicrobial properties only work when bacteria contact their surfaces, making it difficult to kill bacteria in the surrounding infected tissue, and for many polypeptides their antimicrobial properties are reduced or even lost under physiological salt and serum conditions.

Silver nanoparticles (AgNPs) are typical representatives of inorganic antibacterial agents, have the characteristics of broad-spectrum antibacterial, powerful and lasting sterilization, permeation sterilization, promotion of wound repair and regeneration, low drug resistance and the like due to small-size effect, quantum effect and extremely large specific surface area, and are widely applied to surface coatings of titanium implants. After the nano silver is coated on the surface of the titanium implant, the titanium implant has good antibacterial effect on streptococcus mutans, porphyromonas gingivalis, candida albicans and other bacteria. The nano silver has high toxicity to prokaryotic cells such as bacteria and the like and good compatibility with eukaryotic cells, so that the silver has excellent antibacterial performance and low cytotoxicity to organisms, and the cytotoxicity is not observed under a certain dosage.

However, the nano silver is easy to agglomerate, the bactericidal performance after agglomeration is greatly reduced, the technology for coating the nano silver on the surface of the titanium implant is complicated at present, and the antibacterial property of the nano silver is also reduced after coating or surface treatment. More importantly, due to its wide clinical application, multiple strains of silver-resistant bacteria have been isolated

Therefore, there is a need to develop combination drug therapies that are antibacterial by different mechanisms.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the defects in the prior art, the invention provides an organic-inorganic antibacterial composite coating on the surface of a medical implant and a preparation method thereof, according to the respective advantages and disadvantages of a passive organic antibacterial agent antibacterial peptide GL13K and an active inorganic antibacterial agent nano-silver with different antibacterial mechanisms, the two are combined to form the composite coating, so that the effects of avoiding disadvantages and complementing advantages can be achieved, and the organic/inorganic composite antibacterial coating with double antibacterial, synergistic antibacterial, low drug resistance and simple and convenient operation is formed.

The technical scheme is as follows: a preparation method of an organic-inorganic antibacterial composite coating on the surface of a medical implant comprises the following preparation steps:

(1) preparing nano silver powder: preparing nano silver powder by reducing silver nitrate by using citrate as a reducing agent and sugar as a stabilizing agent;

(2) preparation of nano silver-antibacterial peptide GL13K self-assembly nanofiber composite solution: adding nano silver powder into borax-sodium hydroxide buffer solution with the pH value of 9.8 to prepare 0.2mg/mL nano silver solution, adding antibacterial peptide GL13K into water, shaking uniformly to prepare 100mg/mL GL13K solution, then adding GL13K solution into the nano silver solution, wherein the volume ratio of GL13K solution to nano silver solution is 1:99, the final concentration of nano silver and GL13K in the obtained mixed solution is 0.2mg/mL and 1mg/mL respectively, storing the mixed solution at 4 ℃ for 4 days, namely, carrying out self-assembly of GL13K, and forming nano silver-antibacterial peptide GL13K self-assembled nanofiber composite solution;

(3) preparing an organic-inorganic antibacterial composite coating: putting the pretreated medical implant into 48-pore plates, adding 250 mu L of nano silver-antibacterial peptide GL13K compound solution into each pore plate, sealing the edge of each pore plate by using a sealing film, culturing at 4 ℃ for 12h, taking out the medical implant from the solution, carefully flushing with deionized water, and drying with nitrogen to form an organic-inorganic antibacterial composite coating on the surface of the medical implant.

The specific preparation method of the nano silver powder in the step (1) is as follows: dropwise adding 250mM citric acid aqueous solution into 1mM silver nitrate aqueous solution, wherein the volume ratio of the citric acid solution to the silver nitrate solution is 1:50, stirring for 10min in a water bath at 93-94 ℃, centrifuging for 30min under the centrifugal force of 35280RCF, and repeatedly centrifuging for three times to collect precipitated nano-silver; then, dissolving the collected nano silver in deionized water again, centrifuging for 10min under the centrifugal force of 2380RCF, and collecting the precipitated nano silver by repeating the centrifugation for three times; diluting the finally collected nano silver precipitate with deionized water, and measuring Optical Density (OD) value with spectrophotometer until the precipitate is diluted to OD₄₂₀Adding 0.1g of sucrose into each mL of the silver powder 2.9, mixing and stirring for 2h, drying for 24h in a freeze dryer to obtain the nano silver powder, sealing the nano silver powder and storing the nano silver powder at-20 ℃ for later use.

In the step (1), the citrate is trisodium citrate.

The borax-sodium hydroxide buffer solution in the step (2) is prepared from 0.025M borax water solution and 0.1M NaOH water solution.

The pretreatment method of the implant for traditional Chinese medicine in the step (3) is as follows: soaking the medical implant in acetone for 10min, ultrasonically cleaning with deionized water for 15min, ultrasonically treating with cyclohexane for 15min, and blow-drying with nitrogen; then the medical implant is put into a test tube, 5mL of NaOH aqueous solution with the concentration of 5M is added into the test tube, the test tube is covered and fixed, the test tube is placed in a 60 ℃ incubator for alkaline etching for 12 hours, then the medical implant is washed by deionized water and acetone in sequence, and then dried by nitrogen for standby.

The medical implant is a Ti6Al4V micro-implant.

The medical implant surface organic-inorganic antibacterial composite coating prepared by the preparation method is provided.

The organic antibacterial peptide and the inorganic nano-silver adopted by the invention have different antibacterial action mechanisms, and double coatings of the organic antibacterial peptide and the inorganic nano-silver can be considered, but the antibacterial peptide and the nano-silver have completely different coating processes, the antibacterial peptide usually needs to be grafted to the titanium-based surface by using chemical covalent bonds to form a stable coating, the nano-silver is usually coated by methods such as evaporation, sputtering or plasma spraying at high temperature, if the antibacterial peptide is coated firstly and then the nano-silver is coated, the antibacterial peptide is denatured at high temperature, and if the antibacterial peptide is coated firstly and then the nano-silver is coated, the antibacterial peptide is difficult to be grafted covalently, so how to coat the two double coatings becomes a difficult point. According to the invention, firstly, antibacterial peptide GL13K is self-assembled into nano-fiber by a physical and chemical method, then nano-silver is combined to the polar end of the antibacterial peptide nano-fiber by electrostatic force, nano-silver load is embedded in the antibacterial peptide to form a GL 13K-nano-silver complex, and the antibacterial peptide nano-fiber and the nano-silver complex can simultaneously form a stable coating on the surface of a titanium base by utilizing the strong adsorption effect of the antibacterial peptide nano-fiber on the titanium base. According to the invention, the passive organic antibacterial agent antibacterial peptide GL13K and the active inorganic antibacterial agent nano-silver which have different antibacterial mechanisms are combined to form the composite coating according to the respective advantages and disadvantages of the two antibacterial mechanisms, so that the effects of avoiding disadvantages and complementing advantages can be achieved, and the organic/inorganic composite antibacterial coating which has double antibacterial, synergistic antibacterial, low drug resistance and simple and convenient operation is formed.

Has the advantages that: the organic-inorganic antibacterial composite coating on the surface of the medical implant and the preparation method thereof provided by the invention have the following beneficial effects: according to the invention, the passive organic antibacterial agent antibacterial peptide GL13K and the active inorganic antibacterial agent nano-silver which have different antibacterial mechanisms are combined to form the composite coating according to the respective advantages and disadvantages of the two antibacterial mechanisms, so that the effects of avoiding disadvantages and complementing advantages can be achieved, and the organic/inorganic composite antibacterial coating which has double antibacterial, synergistic antibacterial, low drug resistance and simple and convenient operation is formed.

Drawings

FIG. 1 is a graph showing the results of measuring the elemental contents of the coating surfaces of eTi, Ag, GL, and Ag-GL groups using an energy spectrum analyzer.

FIG. 2 is a graph of water contact angle measurements on the surfaces of coatings from groups eTi, Ag, GL, and Ag-GL.

FIG. 3 is a graph showing the colony counts on the surface of the tissue sections of eTi group, Ag group, GL group, and Ag-GL group.

FIG. 4 is a surface colony map of eTi, Ag, GL, and Ag-GL tissue sections.

FIG. 5 is a graph showing HE staining results of tissue sections of eTi, Ag, GL, and Ag-GL groups.

Detailed Description

The Medical implants described in the following examples and comparative examples were Ti6Al4V micro-implants, available from Jeil Medical Corp (korea); silver nitrate was purchased from shanghai shenbo chemical ltd (china); trisodium citrate is purchased from west longa science ltd (china); the antibacterial peptide GL13K was purchased from Hefei peptide Biotech limited (China). The rest materials are all the materials which are commonly used in the market.

The borax-sodium hydroxide buffer solution is prepared from 0.025M borax water solution and 0.1M NaOH water solution.

Example 1

The embodiment provides a preparation method of an organic-inorganic antibacterial composite coating on the surface of a medical implant, which comprises the following preparation steps:

(1) preparing nano silver powder: dropwise adding 10mL of 250mM trisodium citrate aqueous solution into 500mL of 1mM silver nitrate aqueous solution, stirring in a water bath at 93-94 ℃ for 10min, centrifuging for 30min under the centrifugal force of 35280RCF, and repeatedly centrifuging for three times to collect precipitated nano-silver; then, dissolving the collected nano silver in deionized water again, centrifuging for 10min under the centrifugal force of 2380RCF, and collecting the precipitated nano silver by repeating the centrifugation for three times; diluting the finally collected nano silver precipitate with deionized water, and measuring Optical Density (OD) value with spectrophotometer until the precipitate is diluted to OD₄₂₀Adding 0.1g of cane sugar into each mL of the silver powder 2.9, mixing and stirring for 2 hours, drying for 24 hours in a freeze dryer to obtain nano silver powder, sealing the nano silver powder and storing the nano silver powder at the temperature of minus 20 ℃ for later use;

(2) preparation of nano silver-antibacterial peptide GL13K self-assembly nanofiber composite solution: adding nano silver powder into borax-sodium hydroxide buffer solution with the pH value of 9.8 to prepare 0.2mg/mL nano silver solution, adding antibacterial peptide GL13K into water, shaking uniformly to prepare 100mg/mL GL13K solution, then adding 10 muL GL13K solution into 990 muL nano silver solution to obtain mixed solution, wherein the final concentration of nano silver and GL13K in the mixed solution is 0.2mg/mL and 1mg/mL respectively, storing the mixed solution at 4 ℃ for 4 days to perform self-assembly of GL13K, and forming nano silver-antibacterial peptide GL13K self-assembled nanofiber composite solution;

(3) preparing an organic-inorganic antibacterial composite coating: sequentially soaking the micro-implant in acetone for 10min, ultrasonically cleaning with deionized water for 15min, ultrasonically treating with cyclohexane for 15min, and blow-drying with nitrogen for later use; then placing the micro-implant into a test tube, adding 5mL of NaOH aqueous solution with the concentration of 5M into the test tube, covering and fixing, placing the test tube into a 60 ℃ incubator for alkaline etching for 12h, sequentially washing the medical implant by deionized water and acetone, drying by nitrogen for later use, then placing the pretreated micro-implant into 48-hole plates, adding 250 mu L of nano-silver-antibacterial peptide GL13K compound solution into each hole plate, sealing the edges of the hole plates by a sealing film, culturing at 4 ℃ for 12h, taking out the micro-implant from the solution, carefully washing by the deionized water, and drying by the nitrogen to form an organic-inorganic antibacterial composite coating on the surface of the micro-implant.

Comparative example 1

Comparative example 1 is where the surface of the micro-implant is not coated with a coating.

Comparative example 2

Comparative example 2 is a micro-implant coated with a nano-silver coating only.

The preparation method of the nano-silver coating comprises the following steps: and (3) putting the pretreated micro-implant into 48-pore plates, adding 250 mu L of 0.2mg/mL nano-silver solution into each pore plate, sealing the edge of each pore plate by using a sealing film, culturing at 4 ℃ for 12h, taking out the medical implant from the solution, carefully flushing with deionized water, and drying with nitrogen to form a nano-silver coating on the surface of the micro-implant.

Here the nanosilver solution was the same as in example 1.

Comparative example 3

Comparative example 3 is a self-assembled nanofiber coating of antimicrobial peptide GL13K only coated on the surface of the micro-implant.

The preparation method of the antibacterial peptide GL13K self-assembled nanofiber coating comprises the following steps: and (3) putting the pretreated micro-implant into 48-pore plates, adding 1mg/mL antibacterial peptide GL13K solution into each pore plate, sealing the edge of each pore plate by using a sealing film, culturing at 4 ℃ for 12 hours, taking out the medical implant from the solution, carefully washing with deionized water, and drying with nitrogen to form an antibacterial peptide GL13K self-assembled nanofiber coating on the surface of the micro-implant.

The antibacterial peptide GL13K solution was prepared as follows: dissolving GL13K in deionized water to prepare 100mg/mL GL13K aqueous solution, adding GL13K aqueous solution into borax-sodium hydroxide buffer solution with the pH value of 9.8 to prepare 1mg/mL antibacterial peptide GL13K solution of GL13K, and storing for 4 days at 4 ℃ to obtain the antibacterial peptide GL13K solution.

The following tests were conducted on the nano-silver-antimicrobial peptide GL13K composite coating on the surface of the micro-implant prepared in example 1 and the performances of comparative examples 1 to 3. Wherein example 1 is a group of Ag-GL, comparative example 1 is a group of eTi, comparative example 2 is a group of Ag, and comparative example 3 is a group of GL.

1. Characterization of the coating

(1) Elemental composition analysis

The results of analyzing the content of the elements on the surface of the micro-implant coating by using an Energy Dispersive Spectrometer (EDS) are shown in fig. 1, and the contents of the main element components are detected as shown in table 1 below. As can be seen from FIG. 1 and Table 1, the Ag group surface contained 3.2% Ag, and the Ag-GL group detected 2.8% Ag, which confirmed the successful loading of nano-silver onto the micro-implant. Group GL and Ag-GL detected 10.3% and 9.8% N, respectively, demonstrating loading of GL13K onto the surface of the micro-implants.

TABLE 1

(2) Hydropathic and hydrophobic assay

Each group of 3 micro-implants was added with deionized Water in the same volume drop by drop at the screw cap, and a Water contact angle image after 10s was taken to measure the contact angle (WCA). As shown in fig. 2, the water contact angle of Ag group was close to that of the alkali-etched titanium implant eTi group, and water droplets dropped on the surface of the nut and spread out, so that almost no water droplets remained on the surface of the nut, and hydrophilicity was exhibited. And the water contact angle of the GL13K and Ag-GL group coatings is obviously larger than that of eTi groups, the water drop can stay on the surface for a long time and is not changed, and the GL13K enables the surface of the micro-implant to show hydrophobicity.

2. In vivo antibacterial property

After anesthesia, skin preparation and disinfection, a 2cm incision is made along the long axis of the femur on the inner side of the knee joints at both sides, the epidermis is cut, the muscle reaches the periosteum, and the bone surface is exposed by blunt separation. Preparing an implant nest with the diameter of 1.2mm at the center of the distal end joint surface of the femur by using a low-speed grinder under the flushing of a large amount of physiological saline, and controlling the rotating speed at 800 r/min. 10 mul 10 concentration is added into the implant pit⁹eTi groups of micro-implants, AgNPs coating group, GL13K coating group and 5 micro-implants of AgNPs-GL13K composite coating group are randomly implanted into femoral implant nests on the left side and the right side of a rat by CFU/mL methicillin-resistant staphylococcus aureus (MRSA), whether the micro-implants are loosened or not is checked after the micro-implants are implanted, layered suture is carried out, and the wound is closed. After 5 days, the rat is killed, the micro-implant is taken out, and a colony counting experiment is carried out; and (3) intercepting the femoral bone specimen by a dental slow machine, carefully removing redundant tissues, putting the femoral bone specimen into 10% neutral formalin solution for fixation for 48 hours, carrying out decalcification hard tissue slicing, and carrying out HE staining observation.

The colony counting experiment results show that the bacteria collected on eTi groups of micro-implants are the most as shown in fig. 3 and 4; the amount of bacteria on the micro-implants of the Ag group and the GL group is less than that collected on the Ti group (p is less than 0.05); the Ag-GL group micro-implant has the least bacteria, has significant difference (p is less than 0.05) compared with the Ag group and the GL group, and has obviously better antibacterial performance on MRSA than other three groups.

The tissue section HE staining result shows that as shown in FIG. 5, after 5 days of bacteria inoculation, the bone tissues around eTi groups of micro-implants were obviously destroyed, and a large amount of inflammatory cells in the marrow cavity were infiltrated, mainly lymphocytes and neutrophils, indicating severe inflammatory reaction; the Ag group has light inflammation, the thread form of the micro-implant can be seen in the marrow cavity, the compact bone in partial area is irregularly absorbed, the thread continuity is interrupted, and the inflammation is obviously reduced compared with the eTi group; a small amount of inflammatory cell infiltration can be seen in the GL group, the thread shape of the micro-implant is distorted, and a small amount of osteoclast and osteoblast can be seen in a partial area; the Ag-GL group has the lightest inflammation, no obvious bone destruction, complete compact bone, clear thread shape in a marrow cavity, and more blue-stained marrow cells in the lower marrow cavity.

While the embodiments of the present invention have been described in detail, those skilled in the art will recognize that the embodiments of the present invention can be practiced without departing from the spirit and scope of the claims.