Method for removing bacterial biofilm on surface of internally fixed titanium implant by electrochemical anodic oxidation method

Abstract

The invention discloses a method for removing a bacterial biofilm on the surface of an internally-fixed titanium implant through an electrochemical anodic oxidation method, and relates to the technical field of medical instruments, the method comprises the following steps: (1) placing the titanium implant with the bacterial biofilm adhered on the surface as an anode in an electrolyte for anodic oxidation treatment; (2) carrying out glycine air sandblasting on the titanium implant; and (3) standing and soaking the titanium implant in deionized water, and then taking out and drying the titanium implant. According to the method, the electrochemical anodic oxidation technology and the sand blasting technology are combined, efficient removal of the biological membrane on the surface of the titanium implant is achieved, and the problem that bacterial biological membranes are not thoroughly removed through an existing technical method is solved.

Description

Technical Field

[0001] The invention relates to the technical field of medical devices, and in particular to a method for removing bacterial biofilm on the surface of an internal fixation titanium implant by an electrochemical anodic oxidation method. Background Art

[0002] The use of orthopedic implants has greatly promoted human health and helped many patients achieve rapid recovery and mobility. Titanium (Ti) and titanium alloys are widely used in the manufacture of orthopedic implants in the healthcare field due to their excellent mechanical properties, chemical stability, high corrosion resistance and good biocompatibility, such as titanium alloy plates and screws used for fracture fixation, and artificial joint prostheses. However, the infectious complications associated with them, collectively referred to as orthopedic device-related infections (ODRIs), may lead to a decline in the quality of life of patients, or even endanger their lives, bring challenges to the medical team, and impose a high economic burden on society. The treatment of orthopedic device-related infections is a great clinical challenge, mainly due to bacterial resistance, the formation of biofilms on the implant surface and in the bone, limited penetration of antibiotics into the infection site, and the presence of abscesses.

[0003] Debridement of implant-retained implants (DAIR) plays an important role in the treatment of ODRIs, especially in the management of acute infections. The positive significance of DAIR is that it provides a treatment option with less trauma, faster recovery, lower cost and reliable efficacy in specific situations, providing patients and medical teams with a solution that takes both efficacy and economy into account. In debridement of implant-retained implants (DAIR), cleaning the biofilm on the surface of non-removable implant components is a key step, because the bacteria in the biofilm have high drug resistance and anti-immune escape ability. The purpose is to remove the infection focus, reduce the bacterial load, and create good conditions for antibiotic treatment. Currently, the commonly used methods in clinical practice include mechanical removal, pulse irrigation, chemical treatment, ultrasound-assisted cleaning, application or soaking of local antimicrobial agents, etc.

[0004] Although these methods are widely used in clinical practice, they still have certain limitations. Specifically, mechanical removal using tools such as scrapers or soft brushes for physical removal is a direct and effective method, but studies have shown that mechanical removal may not be able to completely remove biofilms, especially in tiny pores or complex structures on the surface of implants, where residual bacteria may lead to recurrent infection. Pulse irrigation can assist in removing loose biofilm fragments through high-pressure pulse irrigation, but its effectiveness is limited, especially in the removal of deep biofilms, where excessive pressure may also cause damage to surrounding healthy tissues. Chemical treatment uses antibacterial solutions or enzyme preparations to treat biofilms, which has a certain risk of cytotoxicity and may affect the health of surrounding tissues. In addition, the permeability of chemical reagents is limited and may not completely destroy the deep structure of biofilms. Ultrasound-assisted cleaning destroys biofilm structures through cavitation effects, but its clinical application may be limited by equipment accessibility and operational complexity. In addition, the energy parameters of ultrasound need to be precisely controlled to avoid potential damage to surrounding tissues.

[0005] In general, these methods have their own advantages in removing biofilms, but they also have certain limitations. Therefore, multiple methods are often combined in clinical practice to improve the treatment effect. But overall, the failure rate of DAIR is still high (less than 50%), and one of the main reasons is that the current methods are not thorough in removing bacterial biofilms. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodizing. The method combines electrochemical anodizing and sandblasting technology to achieve efficient removal of biofilm on the surface of titanium implants, solving the problem of incomplete removal of bacterial biofilm by prior art methods.

[0007] The technical solution of the present invention to solve the above technical problem is as follows: a method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodization is provided, comprising the following steps: (1) The titanium implant with bacterial biofilm attached to the surface is placed in an electrolyte as an anode for anodization treatment; (2) performing glycine air blasting on the titanium implant treated in step (1); (3) The titanium implant treated in step (2) is left to soak in deionized water, and then taken out and dried.

[0008] The method of the present invention utilizes electrochemical principles to form a loose titanium dioxide nanotube layer on the implant surface and the bacterial biofilm interface through an anodic oxidation reaction, loosens the adhesion between the bacterial biofilm and the implant surface, and then utilizes the physical impact formed by glycine sandblasting to completely remove the loose titanium dioxide nanotube layer and the bacterial biofilm at the same time. The method can act on the bacterial biofilm in the tiny pores or complex structures on the implant surface, as well as the deep biofilm, and sets low voltage and low current, which will not cause damage to the surrounding healthy tissues. It has both the effect of deep sterilization and biofilm removal, and is safe for application. It has strong clinical transformation potential.

[0009] Furthermore, in step (1), the electrolyte comprises 0.5-3 wt% ammonium fluoride, 20-40 vt% ethylene glycol, 10-30 vt% glycerol, 0.2-0.5 mol / L magnesium sulfate and 0.1-0.3 mol / L sodium chloride, with the remainder being deionized water.

[0010] Furthermore, in step (1), anodization is performed at a voltage of 8-15 V and a current of 5-30 mA for 10-40 min.

[0011] Furthermore, in step (2), the time of glycine air blasting is 10-120 s.

[0012] Furthermore, in step (3), the mixture is allowed to soak for 12-36 hours.

[0013] Furthermore, in step (3), drying is performed at 50-70° C. for 10-15 h.

[0014] The present invention has the following beneficial effects: 1. The electrochemical anodization method has significant advantages in removing biofilm on the implant surface, with the characteristics of simple operation, short oxidation time and low voltage requirement. This method forms an amorphous TiO2 nanotube (TNT) structure on the implant surface through low-voltage electrochemical oxidation, which has the following key characteristics: (1) Amorphous structure is fragile: Amorphous TNT structure is fragile under mechanical stress and can be easily removed by means such as low-intensity sandblasting; (2) Simultaneous removal of biofilm and nanotubes: Since bacterial biofilm is attached to the surface of TNT nanotubes, as the nanotubes are broken and removed, the biofilm can also be completely removed simultaneously, thus improving the cleaning efficiency; (3) Time and energy consumption advantages: Compared with traditional physical or chemical methods, electrochemical anodization can complete the treatment in a short time and requires a lower voltage, which significantly reduces energy consumption and operation difficulty.

[0015] 2. The method of the present invention forms a fragile nanotube structure through low-voltage electrochemical oxidation, and combines sandblasting or other auxiliary means to remove the biofilm and nanotube layer at one time, which not only simplifies the removal process of the biofilm on the implant surface, but also can achieve efficient and thorough cleaning effects without damaging the substrate, providing a new technical approach for the reuse of implants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of anodizing device; Figure 2 The SEM images of different treatment groups in Experimental Example 1; Figure 3 This is the EDS element analysis result diagram of different treatment groups in Experimental Example 1; Figure 4 This is a diagram of the water contact angle analysis results of Test Example 1; Figure 5 This is the surface roughness analysis result diagram of Test Example 1; Figure 6 SEM images of different treatment groups in the in vitro bacterial biofilm experiment; Figure 7 This is the water contact angle analysis result of in vitro bacterial biofilm experiment; Figure 8 This is the surface roughness analysis result of in vitro bacterial biofilm experiment; Fig. 9 The comparison diagram of photoelectron spectrum of titanium sheet surface in each treatment group; Fig.10 This is a comparison chart of the element composition of the titanium sheet surface in each treatment group; Fig.11 The results of the analysis of the residual Staphylococcus aureus on the titanium sheet surface of each treatment group are shown in the figure; Fig.12 The results of fluorescence staining of organic matter residue on the titanium sheet surface of each treatment group; Fig.13 This is the semi-quantitative analysis result of the residual area of ​​bacteria on the titanium sheet surface of each treatment group; Fig.14 The live-dead staining results of BMSCs cultured on the titanium sheet surface of each treatment group; Fig.15 These are the CCK-8 test results on the titanium sheet surface in each treatment group. DETAILED DESCRIPTION

[0017] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0018] Example 1 A method for removing bacterial biofilm on the surface of an internal fixation titanium implant by electrochemical anodization, comprising the following steps: (1) The titanium implant was ultrasonically cleaned in acetone and anhydrous ethanol for 20 min, then dried at 60 °C for 15 h. The titanium sheet was then placed in the electrolyte as an anode and anodized for 20 min at a voltage of 10 V and a current of 12 mA. (2) performing glycine air blasting on the implant treated in step (1) for 30 s; (3) Soaking the implant treated in step (2) in deionized water for 24 h, then taking it out and drying it at 60° C. for 12 h; Wherein, in step (1), the electrolyte has a formula of 0.5% ammonium fluoride, 20 vt% ethylene glycol, 10 vt% glycerol, 0.2 mol / L magnesium sulfate and 0.1 mol / L sodium chloride, and the remainder is deionized water.

[0019] Example 2 A method for removing bacterial biofilm on the surface of an internal fixation titanium implant by electrochemical anodization, comprising the following steps: (1) The implants were ultrasonically cleaned in acetone and anhydrous ethanol for 20 min, then dried at 50 °C for 24 h. The titanium sheet was then placed in the electrolyte as the anode and anodized for 40 min at a voltage of 8 V and a current of 30 mA. (2) performing glycine air blasting on the titanium implant treated in step (1) for 120 s; (3) The titanium implant treated in step (2) was placed in deionized water for 12 h, then taken out and dried at 50° C. for 15 h; Wherein, in step (1), the electrolyte has a formula of 1.5% ammonium fluoride, 30 vt% ethylene glycol, 20 vt% glycerol, 0.35 mol / L magnesium sulfate and 0.2 mol / L sodium chloride, and the remainder is deionized water.

[0020] Example 3 A method for removing bacterial biofilm on the surface of an internal fixation titanium implant by electrochemical anodization, comprising the following steps: (1) The titanium implant was ultrasonically cleaned in anhydrous ethanol for 30 min, then dried at 70 °C for 12 h, and then placed in an electrolyte as an anode for anodization at a voltage of 15 V and a current of 30 mA for 10 min. (2) performing glycine air blasting on the titanium implant treated in step (1) for 40 s; (3) The titanium implant treated in step (2) was immersed in deionized water for 36 h, and then taken out and dried at 70° C. for 10 h; Wherein, in step (1), the electrolyte has a formula of 3% ammonium fluoride, 40 vt% ethylene glycol, 30 vt% glycerol, 0.5 mol / L magnesium sulfate and 0.3 mol / L sodium chloride, and the remainder is deionized water.

[0021] Test Example 1 The titanium sheet was polished with graded metallographic sandpaper (400#, 600# and 800#) to remove the oxide layer and rough materials on the surface of the titanium sheet. The surface of the polished sample changed from dark gray to silvery white with metallic luster, and there were no obvious rough grinding marks on the surface, ensuring that the surface was smooth and without large scratches. Next, the titanium sheet was ultrasonically cleaned with acetone and anhydrous ethanol for 20 min respectively to ensure thorough cleaning. After cleaning, the sample surface was dried with a hair dryer. The titanium sheet was further ultrasonically cleaned with deionized water (UP water) for 20 min to remove possible solvent residues and tiny particles. After cleaning, the sample was placed in an oven at 60°C for drying to prevent uneven oxidation or other experimental errors due to moisture in subsequent processing.

[0022] A total of four experimental groups were set up, and the treatment methods were: no other treatment (blank control group Ti), anodizing the titanium sheet (anodized titanium sheet group TNT), sandblasting the titanium sheet (sandblasting titanium sheet group AA), and anodizing first and then sandblasting (sandblasting + anodizing titanium sheet group TNT+AA).

[0023] Anodizing treatment (TNT): Anodizing experiments are carried out in a standard electrolytic cell, such as Figure 1 As shown. In the experiment, a 250 mL beaker was used as the electrolytic cell container, and the container contained 100 mL of electrolyte. The electrolyte formula included 1 wt% sodium fluoride, 25% ethylene glycol, 20% glycerol, 0.36 mol / L sodium sulfate and 55% UP water. The composition and concentration of the electrolyte were optimized to ensure the formation of a uniform and stable oxide film during the anodization process. The beaker was placed on a magnetic stirrer to maintain uniform mixing and temperature control of the electrolyte. In this experiment, the cathode and anode of the electrolytic cell were both titanium sheets, and the cathode size was 30×40×1 mm 3 , the anode size is 10×10×1 mm 3 The distance between the two is 30 mm. A 1.5×4.0 mm 2The groove is used to ensure good contact between the sample and the anode. The cathode and cathode are connected to a programmable linear DC power supply respectively through wires to form a complete circuit loop. During the electrolysis process, the sample is just immersed in the electrolyte to ensure the stability and uniformity of the electrolysis process. The electrolysis process is carried out in a step-by-step voltage-boosting mode. At the beginning of the experiment, the voltage gradually increased from 0 V and slowly increased at a rate of 1 V / min until a final voltage of 10 V was reached. After the voltage stabilized, the voltage was maintained until the total oxidation time reached 25 min. During this process, the current was maintained at around 12 mA, ensuring constant current conditions during the anodization process.

[0024] Abrasive blasting (AA): The titanium sheet was blasted using a glycine-based air powder abrasive device. According to the manufacturer's recommendations, the "Liquid" and "Power" settings of the device were adjusted to the maximum to ensure maximum efficiency of the blasting process, and the treatment time was 30 seconds to ensure adequate cleaning effect. After the blasting treatment, the titanium sheet samples were immersed in UP water for 24 hours to remove surface residues and powder. Subsequently, the samples were dried in an oven at 60°C for 12 hours.

[0025] (1) Scanning electron microscope (SEM) observation Due to the poor conductivity of titanium sheet materials, all samples in the experimental group and the blank group must be completely dried before SEM observation. Then, the samples were sprayed with gold by vacuum metal spraying for 70 s to improve their conductivity. After spraying with gold, they were observed by scanning electron microscopy (SEM, model Sigma 500) with an acceleration voltage of 10 kV and a working distance of 10 mm to obtain high-resolution surface morphology images. The results are shown in Figure 2. Figure 2 As shown, ad are SEM images of Ti, TNT, AA and TNT+AA treatment groups, respectively.

[0026] Depend on Figure 2 It can be seen that the surface of the pure titanium substrate without any treatment is relatively smooth and has no obvious microstructure; after TNT treatment, an atypical titanium dioxide nanotube structure is formed on the surface of the titanium substrate, and its tube diameter is about 45 nm; after further AA treatment of the titanium dioxide nanotubes formed by anodization, the nanotubes are removed and the surface becomes relatively flat. At the same time, compared with AA alone, there is not much difference between the two.

[0027] (2) EDS element analysis During the SEM observation, the elements of each group of samples were qualitatively and quantitatively analyzed by combining EDS technology to further explore the effect of different surface treatment methods on the element composition of titanium sheets. Figure 3As shown, ad are the EDS spectra of the Ti, TNT, AA and TNT+AA treatment groups, respectively.

[0028] Depend on Figure 3 It can be seen that the Ti element accounts for 87.07% in the Ti group, and the O element accounts for only 4.56%, indicating that the untreated pure titanium substrate is mainly composed of titanium and the degree of surface oxidation is low; the Ti element content in the AA group decreased to 72.81%, and the O element increased significantly to 22.31%, indicating that sandblasting introduced a certain surface oxidation phenomenon, which increased the proportion of surface oxygen elements; the Ti element content in the TNT group further decreased to 37.99%, and the O element content increased significantly to 49.10%, and a small amount of F element (4.96%) was also detected, proving that the TNT treatment successfully generated a TiO2 nanotube structure with rich oxides on the surface, and the F element may come from the residue of the electrolyte; the Ti element content in the TNT+AA group recovered to 88.31%, close to the level of the pure titanium group, the O element content dropped to 6.04%, and the F element was basically removed, indicating that sandblasting can effectively remove the TiO2 nanotubes generated by anodization, and the surface is restored to a state close to pure titanium.

[0029] (3) Water contact angle The contact angle of water on the titanium sheet surface of each treatment group was measured using a contact angle meter (model FCA2000A3E). The sample was placed on the measuring platform, water was slowly dripped onto the sample surface, and an instantaneous image of the water droplet contacting the surface was taken to calculate the contact angle of the water droplet. This experiment was used to evaluate the hydrophilicity characteristics of the samples with different treatments. The results are shown in Figure 2. Figure 4 As shown, a is a schematic diagram of the water contact angle of each treatment group, and b is a quantitative analysis diagram of the water contact angle of each treatment group.

[0030] Depend on Figure 4It can be seen that the water contact angle of the Ti group surface is 59.61±0.48°, showing a relatively high hydrophobicity. The AA group is similar to the Ti group, and the water contact angle is also 55.48±0.69°, indicating that the surface hydrophilicity has not been significantly changed after AA treatment. Compared with the Ti and AA groups, the TNT surface shows significant hydrophilicity, and the contact angle is significantly reduced, showing a superhydrophilic state close to 0°, which may be related to the nanotube structure formed on the TNT surface. The increase in nanostructures usually increases the contact area between the surface and water, thereby significantly improving the hydrophilicity. In the TNT+AA group, the surface water contact angle rebounded to 46.82±0.34°, which was significantly higher than the TNT group, but still lower than the Ti group and AA group (P<0.001), indicating that the AA treatment introduced a certain degree of hydrophobicity on the TNT surface, but did not completely restore to the original state of Ti. The results of statistical analysis showed that there were highly significant differences between the TNT group and the Ti and AA groups (P<0.001), while the water contact angle of the TNT+AA group was significantly increased compared with the TNT group (P<0.001), but there was still a significant difference compared with the Ti group (P<0.001). In summary, TNT treatment significantly improved the surface hydrophilicity, while TNT+AA treatment restored the surface hydrophobicity to a certain extent. These results show that the hydrophilicity of the material can be regulated by different surface modification methods, thereby affecting its performance in the biological interface.

[0031] (4) Surface roughness The surface roughness (Ra value) of titanium in each treatment group was measured using a surface roughness tester (Perthometer M1, Mahr, Germany) with a diamond probe (tip radius 5 mm). The surface roughness of each sample was measured under the condition of setting a cutoff value of 0.8 mm, and each group of samples was tested three times to ensure the reliability of the results. Finally, the average Ra value of each group of samples was calculated to evaluate the effect of different surface treatments on surface roughness. The results are shown in Figure 2. Figure 5 shown.

[0032] Depend on Figure 5It can be seen that different treatment methods have a significant effect on the surface roughness of Ti. The surface Ra value of the Ti group was 0.23 μm, and the roughness was relatively low. After AA treatment, the Ra value increased to 0.35 μm, which was significantly higher than that of the Ti group, indicating that AA treatment increased the microscopic roughness of the surface. Furthermore, TNT treatment increased the Ra value to 0.44 μm, reaching the highest value. It is speculated that this change is related to the nanotube structure formed on the surface after TNT treatment, thereby increasing the surface roughness. The Ra value of the TNT+AA group was 0.27 μm, which was slightly higher than that of the Ti group, but significantly lower than that of the AA and TNT groups, indicating that after anodizing and sandblasting, the nanotubes on the substrate surface were removed and the surface morphology was close to that of the Ti group. The statistical analysis results showed that there were significant differences between the TNT group and the Ti group, the AA group and the Ti group, and the TNT+AA group and the TNT group (P<0.001).

[0033] Test Example 2 In vitro bacterial biofilm experiment The titanium sheet was cleaned and dried according to Experimental Example 1, and then sterilized in a high pressure steam sterilizer with a pressure of 103.4 kPa (1.05 kg / cm 2 ), the temperature was 121.3℃, and the sterilization time was 1 h. After sterilization, the sample was transferred to a vacuum drying oven and dried for 1 h, and then placed on a clean bench for use.

[0034] The standard strain of Staphylococcus aureus (ATCC 25923) was used for bacterial biofilm culture in this experiment. First, the bacteria were inoculated onto TSB agar plates and cultured at 37°C for 24 h. Then, a single bacterial colony was picked and transferred to a test tube containing 12 mL of TSB medium. After oscillation and mixing, the test tube was placed in a constant humidity shaker (37°C, 150 r / min) and cultured for 17 h until the bacteria were in the logarithmic growth phase. The concentration of the bacterial solution was adjusted to 1×10 7 CFU / mL, as the experimental bacterial solution, was further cultured on the surface of the titanium sheet to form a bacterial biofilm.

[0035] Four experimental groups were set up to test the effects of different biofilm treatment methods, including no other treatment (Sa biofilms), anodizing the titanium sheet (anodized titanium sheet group TNT), sandblasting the titanium sheet (sandblasted titanium sheet group AA), and anodizing first and then sandblasting (sandblasting + anodized titanium sheet group TNT+AA). The treatment process and test methods refer to Experimental Example 1.

[0036] (1) SEM observation results of in vitro bacterial biofilm experiments are as follows Figure 6 As shown, ad are SEM images of S. abiofilms, TNT, AA and TNT+AA treatment groups, respectively.

[0037] Depend on Figure 6 It can be seen that in the untreated biofilm (Sa biofilms group), the Ti surface was completely covered by a thick layer of Staphylococcus aureus biofilm, the bacteria were spherical, closely distributed, and did not show any exposure of the surface structure; after AA treatment, the biofilm on the Ti surface was partially removed, but some biofilm residues could still be seen, indicating that the treatment effect of AA was limited and the biofilm could not be completely removed; after TNT treatment, it can be observed that the structure of the biofilm was destroyed, and some areas showed obvious residues, and the nanotubes (TNT structure) exposed under the biofilm could be vaguely seen; after TNT+AA combined treatment, the biofilm on the Ti surface was almost completely removed, and the surface showed a clean and smooth feature, with no obvious biofilm residue. These results illustrate the significant effect of the combined application of treatment methods on the removal of biofilms, especially the combined treatment of TNT+AA can remove biofilms to the greatest extent.

[0038] (2) The water contact angle test results of in vitro bacterial biofilm experiments are as follows Figure 7 As shown, wherein a is a schematic diagram of the water contact angle of each treatment group, and b is a quantitative analysis diagram of the water contact angle of each treatment group.

[0039] Depend on Figure 7 It can be seen that the water contact angles of Ti and TNT+AA surfaces are 59.61±0.48° and 55.81±1.31°, respectively, and no significant difference is observed between the two, indicating that after TNT+AA treatment, the nanotube structure on the substrate surface has been removed, resulting in similar surface morphology and thus similar water contact angles. TNT treatment often increases the hydrophilicity of the surface by introducing nanoscale pores and surface roughness, while TNT+AA treatment may further remove these nanotubes and restore a flatter surface structure, making the water contact angles of the two surfaces similar. In comparison, the water contact angles of Sa biofilms and AA surfaces are 87.27±5.88° and 94.54±3.8°, respectively, which are significantly higher than those of Ti and TNT+AA surfaces, and show significant hydrophobicity. This result indicates that the interaction between the biofilms formed on these surfaces and water molecules is weak, resulting in an increase in the contact angle and showing hydrophobic characteristics. The hydrophobicity of biofilms is often closely related to the formation of bacterial exopolymers, which reduce the surface affinity for water to a certain extent, resulting in higher contact angles.

[0040] (3) Surface roughness test results of in vitro bacterial biofilm experiments are as follows Figure 8 shown.

[0041] Depend on Figure 8It can be seen that the average Ra value of the Ti surface is 0.23 μm, showing a low surface roughness. Compared with the Ti group, the Ra values ​​of the Sa biofilms, AA and TNT treatment groups increased significantly, 0.4 μm, 0.433 μm and 0.431 μm, respectively, indicating that the surface roughness increased significantly under these treatments. This may be related to the deposition of biofilms and changes in surface microstructures. In particular, under the action of biofilms, the attachment and growth of bacteria may prompt them to form a relatively rough surface, resulting in an increase in Ra values. AA and TNT treatments also significantly increased the roughness due to the introduction of different microstructures on the surface. In particular, TNT treatment may further increase the surface roughness by forming nanotube structures. However, the Ra value of the surface treated with TNT+AA was 0.27 μm, which was slightly higher than that of the Ti group, but significantly lower than that of the Sa biofilms, AA and TNT groups. This result shows that the surface treated with TNT+AA has been anodized and sandblasted, but the nanotubes and biofilm on the substrate surface have been removed, and the surface morphology is close to that of the Ti group, resulting in no significant difference in roughness between the Ti group and the TNT+AA group. Statistical analysis results showed that there was a significant difference between the Ti group and the TNT+AA group (P<0.01).

[0042] (4) XPS tests were performed on different treatment groups. The photoelectron energy spectrum and element analysis results are shown in Figure 2. Fig. 9 and Fig.10 shown.

[0043] Depend on Figure 9-10 It can be seen that the contents of C and N elements on the surface of Staphylococcus aureus biofilm after cultivation were 61.19% and 12.44%, respectively. This result shows that the biofilm is mainly composed of carbon-based and nitrogen-based organic substances, which may be derived from biofilm components such as bacterial extracellular polymers, proteins and nucleic acids. After AA, TNT and TNT+AA treatment, the contents of C and N elements on the surface decreased significantly, indicating that these treatments effectively destroyed the structure of the biofilm or reduced bacterial attachment, thereby leading to the removal of organic components in the biofilm. Specifically, AA and TNT treatments may reduce the adhesion of bacteria and biofilms through changes in surface roughness and the formation of an oxide layer, while TNT+AA treatment may further remove or destroy organic substances attached to the surface through the synergistic effect of the two mechanisms. It is worth noting that after TNT+AA treatment, the contents of Ti and O elements increased significantly, indicating that the surface oxidation treatment promoted the formation of a titanium oxide layer, thereby increasing the surface oxygen content. Meanwhile, the content of Ti element also increased, which may be due to the removal of part of the biofilm or the change of surface material after AA and TNT treatment, exposing more substrate materials.

[0044] (5) Live and dead staining of bacteria SYTO dye can specifically label live bacterial cells, while PI dye can label dead bacterial cells, thereby distinguishing live cells from dead cells. The SYTO / PI dual dye kit was used to stain the washed Staphylococcus aureus biofilms in each treatment group to evaluate the effects of different surface treatments on the formation and removal of bacterial biofilms. Fig.11 As shown, a is the live-dead staining image of Staphylococcus aureus on the surface of titanium sheet in different treatment groups, and b is the semi-quantitative analysis result of the residual area of ​​bacteria.

[0045] Depend on Fig.11 It can be seen that the titanium substrate (Ti group) was almost completely covered, indicating that the Staphylococcus aureus biofilm formed a high degree of attachment and aggregation on the untreated titanium surface. The staining results further showed that although the bacterial cell activity on the titanium surface was different, most of the bacteria survived on this surface and formed a dense biofilm. After AA and TNT treatment, the Staphylococcus aureus biofilm was obviously destroyed, a large number of bacteria died, and the number of live bacteria was significantly reduced. Despite this, a certain number of bacteria remained on the titanium substrate surface after AA and TNT treatment, and the residual area was about 5.1% and 6.4%, respectively. This phenomenon shows that although AA and TNT treatment destroyed the biofilm and killed bacteria to a certain extent, these treatments did not completely remove the bacteria and biofilm residues on the surface. It may be due to changes in the surface microstructure or that the bacterial cells are still partially attached to the surface after treatment, resulting in residues. However, there is almost no bacterial residue on the surface after the combined treatment of TNT+AA. It can be observed by SYTO / PI staining that after this treatment, the biofilm on the surface is almost completely removed, and the residual area is reduced to about 0.14%. This significant difference indicated that the combined TNT+AA treatment exhibited higher efficiency in removing biofilm and almost completely eliminated bacterial residues.

[0046] (6) Fluorescent staining of bacteria In order to evaluate the effect of different surface treatments on biofilm removal, three markers, FITC-protein, DAPI-nucleic acid and ConA-carbohydrate, were further used to reflect the residual protein, nucleic acid and carbohydrate in the biofilm, respectively. Through these markers, the composition and removal degree of the biofilm can be fully reflected.

[0047] After the biofilm was formed on the titanium sheet surface, the non-adherent bacteria were removed by washing with PBS. The biofilm was stained using a micro-modified CLSM method. First, the proteins in the biofilm were stained with 10 μg / mL fluorescein isothiocyanate isomer I (FITC) for 60 min; then, carbohydrates were stained with 0.1 μg / mL Concanavalin A-alexa Fluor 594 conjugate (Con A) for 20 min; finally, nucleic acids were stained with 1 mg / L 4,6-diamino-2-phenylindole (DAPI) for 40 min. After each staining step, the samples were washed three times with PBS to remove excess dye. All staining processes were performed under light-protected conditions. The stained biofilm was dried at room temperature for 15 min and imaged using a confocal laser scanning microscope. The excitation laser wavelengths of the dyes FITC, Con A, and DAPI were 495 nm, 590 nm, and 358 nm, respectively. Combined with the imaging before and after decontamination, the effects of different surface treatments on the biofilm were analyzed. Fluorescence staining images and semi-quantitative results of residual area are shown in Figure 2. Fig.12 and Fig.13 shown.

[0048] Depend on Figure 12-13 It can be seen that after AA and TNT treatment, although the surface morphology has changed, a considerable amount of organic components of the biofilm still remain on the surface. In the AA treatment group, the residual area of ​​the organic components of the biofilm was about 5%, while in the TNT treatment group, the residual area was slightly higher, at 17%. This shows that although AA and TNT treatments have changed the surface properties to a certain extent, they have not been able to completely remove the biofilm, and there are still relatively more bacterial exosecretions and organic components remaining on the surface. In contrast, after the combined treatment of TNT+AA, the biofilm on the surface was almost completely removed. Through the staining results of FITC, DAPI and ConA markers, it can be observed that the organic components of the biofilm almost completely disappeared, and the residual area was only about 0.2%. This result shows that TNT+AA treatment shows significant advantages in removing biofilms.

[0049] (7) BMSCs cell experiment Rat bone marrow mesenchymal stem cells (BMSCs) were extracted by whole bone marrow adherence method, and then subcultured. The third to fifth generations of cells were used for subsequent experiments. After culturing BMSCs cells on the surface of samples in each treatment group for 1, 3 and 5 days, live and dead cell staining and CCK-8 assay were performed. The results were as follows: Fig.14 and Fig.15 shown.

[0050] Depend on Figure 14-15It can be seen that the cell density of BMSCs cultured on Ti and TNT+AA samples is not much different, and the cell morphology is more stretched; while the cell density of Sa biofilms, AA and TNT groups is significantly lower than that of the above two groups, among which the cell morphology of S.abiofilms group is more contracted and not stretched. The cell activity of TNT+AA group is slightly lower than that of Ti group, and the difference between the two is not significant, while the cell activity of Sa biofilms group is significantly lower than that of other groups. After 3 and 5 days of culture, it can be seen that the cell activity of Ti group and TNT+AA group is not significantly different, and the cell activity of Sa biofilms group gradually decreases, which can explain that there is no bacteria on the titanium substrate surface after TNT+AA treatment, so when co-cultured with BMSC, the cell activity is not much different from that of Ti group.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodization, characterized in that: The following steps are involved: (1) The titanium implant with bacterial biofilm attached to the surface is placed in an electrolyte as an anode for anodization treatment; (2) performing glycine air blasting on the titanium implant treated in step (1); (3) The titanium implant treated in step (2) is left to soak in deionized water, and then taken out and dried.

2. The method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodization as claimed in claim 1, characterized in that: In step (1), the electrolyte comprises 0.5-3 wt% ammonium fluoride, 20-40 vt% ethylene glycol, 10-30 vt% glycerol, 0.2-0.5 mol / L magnesium sulfate and 0.1-0.3 mol / L sodium chloride, with the remainder being deionized water.

3. The method for removing bacterial biofilm on the surface of internal fixation titanium implant by electrochemical anodization as claimed in claim 1, characterized in that: In step (1), anodization is performed at a voltage of 8-15 V and a current of 5-30 mA for 10-40 min.

4. The method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodization as claimed in claim 1, characterized in that: In step (2), the time of glycine air blasting is 20-120 s.

5. The method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodization as claimed in claim 1, characterized in that: In step (3), let it soak for 12-36 hours.

6. The method for removing bacterial biofilm on the surface of internal fixation titanium implants by electrochemical anodization as claimed in claim 1, characterized in that: In step (3), dry at 50-70°C for 10-15 h.

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