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Back to Journal »International Journal of Nanomedicine» Volume 16
Authors: Guo Jie, Cao Ge, Wang X, Tang Wei, Di Wuwei, Yan Min, Yang Min, Bi Li, Han Yan
Published on October 27, 2021, the 2021 volume: 16 pages 7249-7268
Single anonymous peer review
Editor approved for publication: Dr. Phong A Tran
Guo Jianbin,1,2,*Cao Guihua,3,*Wang Xing,1 Wen Haotang,1 Weilong Diwu,1 Ming Yan,1 Yang Min,1 Long Bi,1 Han Yisheng 1 1 Department of Orthopedics, The First Affiliated Hospital of Aviation Xi'an, People's Republic of China Army Military Medical University; 2 Department of Joint Surgery, Honghui Hospital, Xi’an Jiaotong University School of Medicine, Xi’an; 3 Department of Geriatrics, First Affiliated Hospital of Air Force Military Medical University, Xi’an, People’s Republic of China * The above authors have contributed equally to this work Corresponding author: Han Yisheng; Long Bi Email [email protected]; [Email protection] Introduction: With the increase in implant infections, finding antibacterial and biofilm coatings has become a new interest for orthopedic surgeons and dentists. In recent years, graphene oxide (GO) has been widely studied due to its excellent antibacterial properties. However, most of these studies focus on solutions, and there are few antibacterial studies on metal surfaces, especially the surface of cobalt-chromium-molybdenum (CoCrMo) alloys. As a new type of food preservative, ϵ-Poly-L-lysine (ϵ-PLL) has a variety of antibacterial activities; however, its antibacterial activity after coating on the surface of the implant is still unclear. Method: In this study, the two-step electrodeposition method was used for the first time to coat GO and ϵ-PLL on the surface of CoCrMo alloy. Then studied its antibacterial and anti-biofilm properties against Staphylococcus aureus and Escherichia coli. Results: The results showed that the formation of bacteria and biofilms on the coating surface was significantly inhibited. The GO and ϵ-PLL composite coatings had the best antibacterial and anti-biofilm effects, followed by ϵ-PLL and GO coatings. In terms of classification, the coating is an anti-adhesion and contact kill/inhibition surface. In addition to oxidative stress, physical damage to GO and electrostatic penetration of ϵ-PLL are the main antibacterial and anti-biofilm mechanisms. Discussion: This is the first study of successfully preparing GO and ϵ-PLL coatings on the surface of CoCrMo alloy by electrodeposition. It provides a promising new method to solve the problem of implant infection in orthopedics and stomatology. Keywords: graphene oxide, ϵ-poly-L-lysine, antibacterial, anti-biofilm, electroplating
The rapid increase in infections caused by bacteria and their biofilms has become one of the greatest potential threats to human health. Antibiotics have been a traditional weapon in the treatment of infections for many years, but the emergence of multidrug resistance (MDR) has made the situation worse. MDR is not just a national issue, but a very complex global phenomenon. 1 Biofilms play an important role in infections caused by MDR bacteria: they are surface attachment communities formed by microorganisms. 2 It produces an extracellular matrix that protects microorganisms from drugs and the host. 3 Therefore, an effective way to combat MDR bacteria and biofilms is to develop new effective antibiotics and/or therapies. 4,5 However, it is difficult to find new effective antibiotics to replace and/or supplement existing antibiotics. 6 In recent years, various materials such as polymers, 7,8 hydrogels, 9,10 antimicrobial peptides (AMPs), 11,12 inorganic nanomaterials13,14 and other materials have been extensively studied.
For plastic surgeons and dentists, implant infections are usually catastrophic. They are the result of improper disinfection and/or improper material handling that lead to bacterial growth and the formation of biofilms on the surface of the material. Medical implant infections caused by bacterial biofilms are a major clinical problem. 3 Reducing implant infections has always been a major challenge faced by plastic surgery and dentists.
In recent years, more and more researchers have begun to coat artificial implants to prevent infection. Coating materials can generally be divided into two categories: one is the combination of antibiotics and polymers, and the other is the type that relies on the intrinsic properties of the material to kill or repel bacteria on the surface. 15 The antibacterial properties of the material surface can be divided into four categories: anti-stick surface, contact kill/inhibition surface, release kill/inhibit surface and remote control sterilization surface.
Cobalt chromium molybdenum (CoCrMo) alloy has become one of the main materials for artificial joints due to its excellent mechanical properties, wear resistance, corrosion resistance and good biocompatibility. 16,17 In recent years, more and more researches have been conducted on the surface modification of CoCrMo alloys. However, most of these studies focus on biocompatibility and mechanical properties, and there are few studies on its antibacterial and anti-biofilm modification. 18-20
With the rapid development of nanoscience and nanotechnology, various antibacterial nanomaterials, such as metal nanoparticles, 21 carbon nanotubes, 22 graphene and its derivatives 23, 24 have been extensively studied. Graphene oxide (GO) is a graphene with abundant oxygen bonds at the edges and defect locations, such as carboxyl (-COOH), carbonyl (-C=O) and hydroxyl (OH) on the inlet side. GO was first used in medical research as a drug delivery agent in 2008. 25 However, since the antibacterial activity of GO was first discovered in 2010, 26 its antibacterial properties have attracted widespread attention in medical research.
ε-Poly-L-Lysine (ε-PLL) is a natural polymer synthesized by microorganisms, which is characterized by the formation of peptide bonds between the carboxyl group of lysine and the ε-amino group. 27 It is widely used in various food, medicine, and electronic products. 28 ε-PLL has been found to have broad-spectrum antibacterial activity, 29 has a minimum inhibitory concentration of less than 100 µg/mL for bacterial growth, and is easily soluble in water, biodegradable, stable at high temperatures, acidic and alkaline conditions. 27
In this study, we used electrodeposition to coat GO and ε-PLL on the surface of CoCrMo alloy for the first time. Then we characterized the coating and tested the mechanical properties and hydrophilicity and hydrophobicity of the coating surface. We further compared the antibacterial and anti-biofilm effects of each coating on Staphylococcus aureus and Escherichia coli, hoping to provide a new direction for the clinical treatment of CoCrMo alloy prosthesis surface infection.
Medical grade CoCrMo alloy (Northwest Nonferrous Metal Research Institute, Xi'an, China Φ=12mm, δ=2mm) is polished to mirror level with mechanical polishing and diamond paste. Before each experiment, the CoCrMo alloy plate was ultrasonically cleaned for 20 minutes in the sequence of deionized water-absolute ethanol-acetone-deionized water, and then dried with N2 flow.
Use a GO solution with a concentration of 100 µg/mL. The alloy sheet is placed on the anode as the working electrode, and the graphite electrode is placed on the cathode. The speed of the magnetic stirrer is 500 RPM. The voltage of the DC stabilized power supply increases to 20 V at a rate of 1 V per second for 10 minutes. 30 After the reaction, the sample was gently rinsed with deionized water and dried in a stream of N2. The surface of the modified sample is named CoCrMo/GO.
In order to optimize the ε-PLL coating on the CoCrMo surface, an orthogonal experiment table with 3 factors and 4 levels was used to design the experiment. 3 factors and 4 levels are: ε-PLL concentration 0.05 W/V%, 0.1 W/V%, 0.15 W/V% and 0.2 W/V%; voltage: 5 V, 10 V, 15 V and 20 V ; Time: 5 minutes, 10 minutes, 15 minutes and 20 minutes. According to the experimental test table, a total of 16 tests are required (Supplementary Material Table 1). The alloy sheet is placed on the cathode as a working electrode, and the graphite electrode is placed on the anode. The speed of the magnetic stirrer is 500 RPM. The voltage of the DC regulated power supply increases to the corresponding operating voltage at a rate of 1 V per second. After the reaction, the alloy flakes were gently washed with deionized water and dried in a stream of N2. The modified sample surface is expressed as CoCrMo/ε-PLL.
In order to prepare the GO and ε-PLL composite coating on the CoCrMo alloy surface, we first prepared the GO coating according to the method. We again use the orthogonal table of 3 factors and 4 levels of ε-PLL coating (Supplementary Material Table 1). CoCrMo/GO is used as the cathode, and the graphite electrode is used as the anode, immersed in different concentrations of ε-PLL aqueous solution. Electrodeposition is performed at room temperature. The magnetic stirring speed is 500 RPM. After the electrodeposition was completed at different voltages and times, the alloy plates were taken out and dried in a stream of N2 at room temperature. The surface of the modified sample is marked as CoCrMo/GO/ε-PLL.
The surface morphology and morphology of the material were observed by scanning electron microscope (FE-SEM, S-4800, Hitachi High Technologies, Tokyo, Japan) and atomic force microscope (AFM, Agilent 5500 SPM, USA). After coating, each group of samples was mechanically polished again to remove the surface coating, and then cleaned by ultrasonic impact. Then, the composition change of each group of CoCrMo alloy elements was detected by scanning electron microscope energy spectrometer (SEM-EDS). There are 3 samples in each group, 3 points for each sample, and the average value of each element is calculated. A Fourier transform infrared (FTIR) spectrometer (VERTEX70 FT-IR-Spectrometer, Bruker Optics, Germany) with an attenuated total reflection accessory (ATR) was used to analyze the chemical composition of the modified surface. The hydrophilicity and hydrophobicity were measured using a contact angle meter (DSA 30, Kruss, Germany) by dripping 4 µL of ultrapure water onto each sample. There are 6 samples in each group, and 6 different measurement areas are selected for each sample. A nanoindenter (Agilent Nano Indenter G200, USA) was used to measure the mechanical properties of the coating surface. Each group has 3 samples, and each sample measures 3 data points.
Gram-positive Staphylococcus aureus (S. aureus ATCC 25923) and Gram-negative Escherichia coli (E.coli ATCC 25922) were used to evaluate their antibacterial and anti-biofilm activities. These two bacteria were incubated overnight in brain heart infusion (BHI, Solarbio, China) medium. Before each study, the two bacteria were adjusted to 106 CFU/mL using a turbidimeter. All subsequent bacterial cultures are completed in a 0.05% dioxide incubator. All alloy samples were sterilized with Go60 before bacterial culture.
Put four sets of samples in a 24-well plate (each set is in triplicate). Then, add 1 mL of 106 CFU/mL bacterial suspension to each well and incubate for 24 and 48 hours. After that, each group of samples was gently taken out and washed with PBS (pH 7.2) 3 times, and then fixed in 2.5% glutaraldehyde at 4°C overnight. The samples were dehydrated with gradient alcohol for 10 minutes, and each gradient was soaked in hexamethyldisilane for 30 minutes. After drying, the sample was sprayed with gold and observed with FE-SEM.
After the incubation, the samples of each group were taken out and rinsed gently with PBS 3 times to remove unadhered bacteria. The sample was then immersed in physiological saline and vibrated in ultrasonic waves for 10 minutes to separate the bacteria adhering to the surface of the material. Then carry out gradient dilution, take 10 μL of bacterial suspension and evenly spread it on the blood plate medium, and count CFU after 24 or 48 hours of culture. Use the following formula to calculate the antibacterial rate and adhesion.
Use Alamar blue detection kit (Alamar Blue, A7631, Solarbio, China) to evaluate the viability of bacteria on the surface of different materials. After Staphylococcus aureus and Escherichia coli were cultured on different surfaces for 24 hours and 48 hours, respectively, the samples were gently washed with PBS to remove unadhered bacteria. After that, 500 µL of 10% Alamar blue was added to the surface of each sample, and then incubated at 37 °C for 2 hours. After 31, transfer 100 µL of culture medium to a 96-well black plate, and measure the fluorescence intensity (FI) with a fluorescence spectrophotometer (Biotek, Synergy H1, USA) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm.
The FilmTracer Live & Dead Bacterial Backlight Kit (ZeYe Biotechnology Inc, China) was used to detect fluorescent images of Staphylococcus aureus and E. coli with different coatings at 24 and 48 hours. For two bacterial species, Staphylococcus aureus and Escherichia coli, the staining dissolution is prepared by mixing 100 µL DMAO and 200 µL EHD-III in a microcentrifuge tube, and then adding 800 µL 0.85% NaCl solution to mix thoroughly. After the two bacteria were incubated on different surfaces for 24 and 48 hours, 40 µL of stain was dropped on each substrate, and then stained in a dark room at room temperature for 15 minutes. After dyeing, the substrate is gently rinsed with deionized water. A laser scanning confocal microscope (LSCM, Fluoview FV1000, Olympus Corporation, Tokyo, Japan) was used to observe the fluorescence of the bacteria. Use green excitation light (488 nm) to observe live bacteria (complete membrane). Red excitation light (543 nm) is used to observe dead bacteria (damaged membrane).
The biofilm was stained with Concanavalin A fluorescein isocyanate conjugate (ConA-FITC C7642; Sigma-Aldrich Inc, USA) to evaluate the effect of each coating on the biofilm at 24 and 48 hours. 32 After incubating the two bacteria on different surfaces, rinse gently with PBS 3 times. Then add 1 mL of 2.5% glutaraldehyde to each well, place in a refrigerator at 4 ℃ for 1.5 h, and then gently rinse with PBS 3 times. After that, drop 40 µL of ConA-FITC with a concentration of 50 µg/mL on the surface of the substrate and incubate at room temperature for 30 minutes. The bacteria are then observed under LSCM (signal obtained is 488 nm; emission is 552 nm).
The crystal violet (CV) assay was used to quantify the biofilm formation of Staphylococcus aureus and E. coli. After culturing the bacteria to the corresponding time point according to the above method, gently wash off the unadhered bacteria on the surface of the material with PBS. Then, fix the biofilm with 2% formalin at room temperature for 15 minutes. Then put the sample into a new 24-well plate, add 1 mL of 0.1% CV stain (Sigma-Aldrich, USA) to each well for 5 minutes, and then wash with PBS 3 times to remove excess stain. Then add 1 mL of 95% ethanol to each well and incubate for 15 minutes. After incubation, transfer 200 µL of ethanol to a 96-well plate, and measure the absorbance at 570 nm using a spectrophotometer (Biotek, Synergy H1, USA).
In order to determine whether the antibacterial effect is affected by the release of certain substances, the method of observing the zone of inhibition is used. Mueller-Hinton agar plate (MH plate) is evenly coated with 1.5×108 S. aureus and E.coli, and then each group of materials is gently placed on the MH plate and cultured in a 0.05% nitrogen dioxide incubator. Continuously observe the formation of bacteriostatic zone in each group.
To study the level of ROS in bacterial cells, an intracellular ROS detection kit (Beyotime, China) was used according to the manufacturer's instructions. As in the previous experiment, Staphylococcus aureus and Escherichia coli were cultured on different substrate surfaces for 24 hours and 48 hours to gently wash off bacteria that did not adhere to the surface. Transfer the sample to a new 24-well plate. Then, add 500 µL DCFH-DA (10 mM) to each well and incubate at room temperature for 30 minutes.
Finally, transfer 100 µL of medium to a 96-well blackboard, and use a microplate reader to detect the fluorescence intensity corresponding to DCF at 485 nm extinction wavelength and 535 nm emission wavelength. 31
Use statistical analysis of variance and Fisher's protected least significant difference (LSD) test to estimate intra- and inter-group differences. Using SPSS 20.0 software for statistical analysis, P<0.05 was considered significant.
The CoCrMo alloy group is the control group, which is denoted as CoCrMo. Our experimental materials are divided into four groups: CoCrMo, CoCrMo/GO, CoCrMo/ε-PLL and CoCrMo/GO/ε-PLL. After coating, the surfaces of different groups of materials show different colors (Figure 1). The CoCrMo alloy sheet is silver-white (Figure 1A). CoCrMo/GO is golden (Figure 1B). The surface of the CoCrMo/ε-PLL group is very similar to the surface of the CoCrMo alloy group, which is also silver-white (Figure 1C). CoCrMo/GO/ε-PLL is dark blue (Figure 1D). Figure 1 Surface appearance after CoCrMo alloy coating. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
Figure 1 Surface appearance after CoCrMo alloy coating. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
After 16 sets of experiments were completed using the parameters of the orthogonal experiment table, the surface morphology of each set of materials was observed with SEM (Figure 2). In CoCrMo/ε-PLL (Figure 2A), the appearance of all groups is different from the control group, as if covered by a film. When the voltage and concentration are low, the coating is in a thin film state, and as the concentration, time and voltage increase, the coating is clustered. In the CoCrMo/GO/ε-PLL group (Figure 2B), it can be seen that there is almost no PLL coating on the surface of some groups (Ep 1). Some groups are arranged in an orderly manner, while others are in a state of chaos (episodes 7, 8, and 10). In order to ensure the uniformity of the subsequent experiments, after we observed the two sets of morphologies, we decided to use experiment 2 (ε-PLL 0.05 W/V%, 10 V 10 min) for follow-up research, and uniformly package the ε-PLL Coated on the surface of CoCrMo and GO, and the surface of GO is not completely blurred. Figure 2 Orthogonal experiment results. (A) ε-PLL coating under different parameters (E1-E16). (B) GO and ε-PLL composite coating under different parameters (Ep 1-Ep 16).
Figure 2 Orthogonal experiment results. (A) ε-PLL coating under different parameters (E1-E16). (B) GO and ε-PLL composite coating under different parameters (Ep 1-Ep 16).
After determining each group of parameters, we observe the surface of each group of materials by SEM at different magnifications (Figure 3). There are obvious scratches on the surface of CoCrMo alloy (yellow arrow). On the CoCrMo/GO group, we can see that GO is symmetrically attached to the surface of the alloy, and GO wrinkles (blue arrows) can be observed on the surface of the alloy. After further modifying CoCrMo with ε-PLL, it can be seen that the surface of CoCrMo is a dense film (red arrow), which is different from the bare CoCrMo alloy. However, on the CoCrMo/GO/ε-PLL group, we can see that GO wrinkles (blue arrow) and ε-PLL (green arrow) are uniformly distributed in the form of particles. Figure 3 SEM observation of CoCrMo, CoCrMo/GO, CoCrMo/ε-PLL and CoCrMo/GO/ε-PLL.
Figure 3 SEM observation of CoCrMo, CoCrMo/GO, CoCrMo/ε-PLL and CoCrMo/GO/ε-PLL.
In order to determine whether the composition of the CoCrMo alloy changes after electroplating, polish and clean each group of samples, and use SEM-EDS to detect the composition changes of each group of CoCrMo alloys (Figure 4). We calculated the mean value of each element in each group and found that the difference between each element and each group was not statistically significant (Table 1, P>0.05). Table 1 Average values of elements in each group (P>0.05) Figure 4 Energy dispersion spectrum of CoCrMo alloy elements. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
Table 1 Mean value of each element in each group (P>0.05)
Figure 4 Energy dispersion spectrum of elements in CoCrMo alloy. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
In order to determine the three-dimensional structure of the material surface before and after the modification, we used AFM to analyze each group of samples (Figure 5). The surface of the CoCrMo alloy is uneven, and scratches produced during the polishing process can be seen. However, after GO and ε-PLL are modified, a uniform organic layer can be seen covering the alloy surface. These scratches became blurred or even disappeared, and wrinkles of GO and ε-PLL particles were observed at the same time. By further comparing the surface roughness of the four groups of materials (9 different 3μm×3μm areas were analyzed for each sample), it was found that the surface roughness of the materials modified with GO and ε-PLL was significantly reduced. The CoCrMo/ε-PLL group has the lowest roughness, and the CoCrMo alloy group has the highest roughness. There was no statistically significant difference between the CoCrMo/GO group and the CoCrMo/GO/ε-PLL group (Figure 6). GO and ε-PLL fill the grooves on the surface of the CoCrMo alloy and compensate for the unevenness of the CoCrMo alloy. Figure 5 The surface morphology and three-dimensional structure of each group of materials. Figure 6 Comparison of four groups of roughness (*P<0.01, **P<0.001).
Figure 5 The surface morphology and three-dimensional structure of each group of materials.
Figure 6 Comparison of four groups of roughness (*P<0.01, **P<0.001).
The wetting characteristics of the four groups of surfaces are shown in Figure 7. Compared with the control group, the contact angles of all experimental groups were significantly reduced (P<0.05). The CoCrMo/ε-PLL group has the best hydrophilicity. The change in wettability is consistent with the presence of the ε-PLL layer, as reported by other authors. 33,34 Figure 7 Comparison of surface hydrophilicity of four groups of materials (*P<0.05, **P<0.01).
Figure 7 Comparison of surface hydrophilicity of four groups of materials (*P<0.05, **P<0.01).
In order to verify the mechanical properties of the coating surface, we use a nanoindenter to measure each group. The elastic modulus and Vickers hardness of the control group were the highest, and the three experimental groups all decreased to varying degrees (Figure 8). We found that there was no statistically significant difference in elastic modulus except for the CoCrMo/GO group. The Vickers hardness results showed that, compared with the CoCrMo group, the difference between the CoCrMo/GO group and the CoCrMo/GO/ε-PLL group was statistically significant, while the difference between the CoCrMo/ε-PLL group was not statistically significant (Table 2). Table 2 Elastic modulus and Vickers hardness value (*P<0.05) Figure 8 Nanoindentation value of each group. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
Table 2 Elastic modulus and Vickers hardness value (*P<0.05)
Figure 8 Nanoindentation value of each group. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
After modification with GO and ε-PLL, each substrate was characterized by FTIR-ATR. The spectrum in Figure 9 shows the different types of oxygen functional groups in CoCrMo/GO. A broad OH stretching peak appears at 3286 cm-1, a strong C=O peak appears at 1627 cm-1, a C-OH stretching peak appears at 1265 cm-1, and a CO stretching peak appears at 1080 cm-1 , It is confirmed that after GO is successfully modified, a large number of hydrophilic groups such as -OH, -COOH and epoxide are formed on the surface of the material. The CoCrMo/ε-PLL group exhibits characteristic PLL absorption characteristics. The C=O stretching vibration peak appears at 1645 cm-1, and the characteristic CH2 stretching vibration appears at 2856 cm-1 and 2923 cm-1. The CoCrMo/GO/ε-PLL group showed both GO and PLL absorption characteristics, CO stretching vibration at 1140 cm-1, C=O stretching vibration at 1649 cm-1, and 2856 cm-1 and 2922 cm-1 CH2 stretching vibration and OH stretching vibration at 3288 cm-1. The results showed that GO and ε-PLL were successfully coated on the surface of CoCrMo alloy. Figure 9 FTIR-ATR spectra of ε-PLL, GO and GO/ε-PLL on CoCrMo alloy.
Figure 9 FTIR-ATR spectra of ε-PLL, GO and GO/ε-PLL on CoCrMo alloy.
Use FE-SEM to observe the morphology of bacteria on each surface. Figure 10 shows the morphological changes of bacteria, illustrating the degree and nature of bacterial cell damage. The surface of CoCrMo alloy shows that Staphylococcus aureus grows well, and the cell morphology is smooth, round and complete (A1, A2). The number of Staphylococcus aureus on the surface of GO was significantly reduced, and some bacterial cell membranes were damaged, and cell perforation and atrophy (B1, B2) were visible. On the surface of CoCrMo/ε-PLL, more Staphylococcus aureus cell membranes ruptured and shrank (C1, C2). The number of bacterial cell deaths in the CoCrMo/GO/ε-PLL group increased significantly, and the cells were severely deformed. In addition to perforation and contraction, they were also accompanied by rupture and bursting (D1, D2). Figure 10 The morphology of Staphylococcus aureus ((A) 24 h, (B) 48 h) and E. coli ((C) 24 h, (D) 48 h) on the surface of each group.
Figure 10 The morphology of Staphylococcus aureus ((A) 24 h, (B) 48 h) and E. coli ((C) 24 h, (D) 48 h) on the surface of each group.
The situation is similar in E. coli. On the surface of CoCrMo, E. coli is short rod-shaped, the cell membrane is intact, and the fimbriae are visible (E1, E2). On the surface of GO, the number of E. coli is significantly reduced. The fimbriae disappeared, and part of the ruptured membrane content was discharged (F1, F2). On the surface of ε-PLL, the number of Escherichia coli is relatively reduced, the pili disappears, and the cells rupture and shrink (G1, G2). On the surface of CoCrMo/GO/ε-PLL, the normal form of E. coli is almost invisible: cell rupture, atrophy, and cell membrane division content can be seen everywhere (H1, H2).
For cells with no obvious morphological changes under the scanning electron microscope, to further determine whether they are dead, the live bacteria/dead bacteria staining method is used. Almost all of the CoCrMo alloy group showed green fluorescence, and the fluorescence intensity at 48 hours was significantly higher than that at 24 hours (Figure 11A1, B1, C1, D1). There are both green and red fluorescence in the CoCrMo/GO group. The intensity of red fluorescence is higher than that of green, and the overall fluorescence intensity is lower than that of the CoCrMo group (Figure 11 A2, B2, C2, D2). Compared with the CoCrMo/GO group, the CoCrMo/PLL group has a lower fluorescence intensity and a higher proportion of red fluorescence (Figure 11 A3, B3, C3, D3). Almost all of the CoCrMo/GO/ε-PLL group showed red fluorescence, and the fluorescence intensity was significantly reduced, indicating that the bacteria adhered very little and the adhered bacteria basically died (Figure 11A4, B4, C4, D4). Figure 11 Fluorescence staining of live and dead bacteria from different coating surfaces. (A) Staphylococcus aureus. (B) Escherichia coli.
Figure 11 Fluorescence staining of live and dead bacteria from different coating surfaces. (A) Staphylococcus aureus. (B) Escherichia coli.
The plate counting method was used to evaluate the antibacterial activity and adhesion rate of Staphylococcus aureus and Escherichia coli. After the bacteria on the surface of the material are fully shaken by ultrasonic vibration, each group of bacteria is diluted 104 times and inoculated on the blood plate medium. The results showed that there was a significant difference between the experimental group and the control group, and the CoCrMo/GO/ε-PLL group had the least colonies (Figure 12). Figure 12 The colonies of each group of Staphylococcus aureus and Escherichia coli on the blood plate.
Figure 12 The colonies of each group of Staphylococcus aureus and Escherichia coli on the blood plate.
By further counting the number of colonies in each group, we found that the relative adhesion rate was the highest in the control group. When the relative adhesion rate of the control group was defined as 100%, the adhesion rate of Staphylococcus aureus in the CoCrMo/GO, CoCrMo/ε-PLL and CoCrMo/GO/ε-PLL groups was 22.8% on the first day, and 22.8% on the second day. 15.35% and 8.86%, as well as 26.6%, 10.8% and 6.37% (Figure 13A). These figures for E. coli on the first day were 25.4%, 20.3%, and 10.6%, respectively, and on the second day were 38.2%, 23.4%, and 12.5% (Figure 13B). Figure 13 Relative adhesion rate and antibacterial rate of each group. The relative adhesion rate of Staphylococcus aureus (A) and Escherichia coli (B) and the antibacterial rate of Staphylococcus aureus (C) and Escherichia coli (D). *And #P<0.001.
Figure 13 Relative adhesion rate and antibacterial rate of each group. The relative adhesion rate of Staphylococcus aureus (A) and Escherichia coli (B) and the antibacterial rate of Staphylococcus aureus (C) and Escherichia coli (D). *And #P<0.001.
In our research on the antibacterial rate, we defined the antibacterial rate of the control group as zero. We found that the CoCrMo/GO/ε-PLL group had the best antibacterial rate. On the first day, the antibacterial rates of the CoCrMo/GO, CoCrMo/ε-PLL and CoCrMo/GO/ε-PLL groups against Staphylococcus aureus were 77.2%, 84.65%, and 91.94%, respectively, and on the second day, they were 73.4%, respectively , 89.2% and 93.63% (Figure 13C). The antibacterial rates in E. coli were 74.6%, 79.7%, and 89.4% on the first day, and 61.8%, 76.6%, and 87.5% on the second day (Figure 13D).
Alama blue staining was used to assess the viability of the bacteria on the surface of each group. The result is shown in Figure 14. The fluorescence intensity of the CoCrMo/GO/ε-PLL group is the lowest, followed by the CoCrMo/ε-PLL and CoCrMo/GO groups. This means that the bacterial activity of the control group is the strongest, while the bacterial activity of each experimental group is weakened, and the weakest is CoCrMo/GO/ε-PLL. The pairwise comparison between each group is also statistically significant (p<0.05). Figure 14 Fluorescence intensity of different samples (* and #P<0.01, ** and ##P<0.001). (A) Staphylococcus aureus. (B) Escherichia coli.
Figure 14 Fluorescence intensity of different samples (* and #P<0.01, ** and ##P<0.001). (A) Staphylococcus aureus. (B) Escherichia coli.
The adhesion of bacteria to the surface of the material is initially reversible, but becomes irreversible when the biofilm is formed. In this study, we observed the structure of the biofilm under SEM (Figure 15). Our research shows that GO and ε-PLL have antibacterial effects on the surface of CoCrMo alloy. In order to further study whether they have anti-biofilm effects, we performed ConA-FITC staining and CV determination. Figure 15 The biofilm structure of Staphylococcus aureus (red arrow) and E. coli (green arrow).
Figure 15 The biofilm structure of Staphylococcus aureus (red arrow) and E. coli (green arrow).
The glycocalyx in the biofilm can be stained by ConA-FITC and emit green fluorescence in the CLSM image. 32 Using this method, biofilms can be observed even under an electron microscope. As seen in Staphylococcus aureus (Figure 16A), a large amount of green fluorescence was observed on the surface of the control group, and the second day was significantly stronger than the first day. The green fluorescence on CoCrMo/GO and CoCrMo/ε-PLL was significantly reduced, and only a small amount of green fluorescence appeared on CoCrMo/GO/ε-PLL on the second day. The same trend was observed in E. coli (Figure 16B). Generally speaking, the fluorescence intensity of Escherichia coli is weaker than that of Staphylococcus aureus, indicating that the ability of Escherichia coli to form a biofilm is lower than that of Staphylococcus aureus. Figure 16 Biofilm staining results. (A) Staphylococcus aureus. (B) Escherichia coli.
Figure 16 Biofilm staining results. (A) Staphylococcus aureus. (B) Escherichia coli.
To further quantify the biofilm produced in each group, we performed a CV measurement. The CV measurement was first described by Christensen in 1985 and has been continuously improved to be applied to the entire microbial biofilm quantification. 35 As described in the results (Figure 17), the absorbance of the control group was significantly higher than that of the other groups. Among the four experimental groups, the CoCrMo/GO/ε-PLL group had the lowest absorbance, while CoCrMo/GO and CoCrMo/ε-PLL There was no significant difference in absorbance between the groups. The overall absorbance of the Escherichia coli group is slightly lower than that of the Staphylococcus aureus group, which also indicates that the ability of Escherichia coli to produce biofilms is poor. Figure 17 CV measurement results (* and #P<0.05, ** and ## P<0.01). (A) Staphylococcus aureus. (B) Escherichia coli.
Figure 17 CV measurement results (* and #P<0.05, ** and ## P<0.01). (A) Staphylococcus aureus. (B) Escherichia coli.
In order to determine whether the experimental group released a certain substance that caused its antibacterial activity, we adopted the method of observing the zone of inhibition. The continuous observation time is 3 days. The results showed that there was no zone of inhibition in both groups (Figure 18). Figure 18 Inhibition zone experiment of each group. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
Figure 18 Inhibition zone experiment of each group. (A) Cobalt Chromium Molybdenum; (B) CoCrMo/GO, (C) CoCrMo/ε-PLL and (D) CoCrMo/GO/ε-PLL.
In order to verify whether ROS affects the antibacterial activity of the material surface, we tested the ROS level of each group. The detection results of ROS levels in each group are shown in Figure 19. The results showed that the ROS level of the CoCrMo/GO/ε-PLL group was significantly higher than the other three groups (P<0.001). However, there was no significant difference in ROS levels between the CoCrMo/ε-PLL and CoCrMo/GO groups. The results show that ROS level is one of the main antibacterial mechanisms of GO and ε-PLL. Figure 19 Fluorescence intensity of each group of DCF (* and #P<0.05, ** and ##P<0.01). (A) Staphylococcus aureus. (B) Escherichia coli.
Figure 19 Fluorescence intensity of each group of DCF (* and #P<0.05, ** and ##P<0.01). (A) Staphylococcus aureus. (B) Escherichia coli.
In the field of joint surgery and dentistry, the appearance of artificial joints and dentures has greatly improved the quality of life of patients. As the population ages, the number of patients receiving joint replacements and dental implants is increasing every year. 36 Infection is a catastrophic complication in orthopedics and dentistry, and is more difficult to manage due to the use of implant components. 37 The mechanism of bacterial adhesion, aggregation on biomaterials and biofilm formation include van der Waals forces, electrostatic forces, hydrophobic forces, and glycoprotein-mediated forces. 37,38 Therefore, the development of materials that reduce the adhesion of pathogenic bacteria to the surface of the prosthesis and the formation of biofilms is 39,40 In recent years, more and more researchers have begun to coat artificial implants to prevent infection.
Through literature review, we know that GO and ε-PLL are both substances that have been proven to have broad-spectrum antibacterial properties. However, most antibacterial research is focused on the solution, and rarely on the alloy surface. GO in aqueous solution is negatively charged 30, and ε-PLL is positively charged, 41 makes it possible to deposit GO and ε-PLL on the alloy surface by electroplating. In this study, GO and ε-PLL were deposited on the surface of CoCrMo alloy by electrodeposition for the first time. As the substrate of the coating, the stability of CoCrMo alloy under voltage is one of the decisive factors in this study. A large number of documents prove that CoCrMo alloy has high biological inertness and has been widely used in artificial joints for many years. Likewise, it is reported that CoCrMo alloy is inherently more biologically inert than TiAlV alloy. 42 One study involved nitriding the surface of CoCrMo alloy to improve its corrosion resistance. The specific method is to place the CoCrMo alloy at 400°C and load the voltage from 500V to 1100V. After 4 hours, no change in the composition of the CoCrMo alloy is observed except for the addition of a nitride layer on the surface. 19 During joint replacement surgery, the surgeon performed metal implants. High voltage and high frequency current are applied near the object to stop the bleeding of adjacent blood vessels, and the voltage is as high as 5 kV. 43 In addition, this study showed only slight damage to the CoCrMo alloy under high-current electrosurgical surgery. Fortunately, in these reports, no changes in alloy composition were found after high pressure treatment. In our current study, only a voltage of 20V is used for electrodeposition. Therefore, it is impossible to change the ion dissolution state of this alloy at such a low voltage. In order to further confirm our statement, we performed ultrasonic vibration cleaning and mechanical polishing again on the material after the voltage was applied, and the alloy composition was found to be unchanged through SEM-EDS scanning.
After successful coating, the in vitro antibacterial and anti-biofilm activities of GO and ε-PLL on the surface of CoCrMo alloy were systematically studied. We use SEM to observe cell morphology, live/dead staining, colony count, Alamar blue staining and other methods to study the antibacterial properties of each group of materials. The results showed that GO and ε-PLL showed a significant inhibitory effect on Staphylococcus aureus and Escherichia coli on the CoCrMo alloy surface, resulting in bacterial cell aggregation and decreased activity. In the CoCrMo/GO/ε-PLL group, perforation, shrinkage, atrophy, rupture and cell membrane division of bacterial cells can be seen everywhere.
Biofilm formation is one of the most important events in the development of biological material infections, and it is also a clinical problem. 3 Generally speaking, it has gone through several stages, including bacterial community development, maturation and decomposition. 37 implantation and difficult to reverse, because traditional treatment methods usually cannot effectively treat biofilms. 44,45 Therefore, it is very important to prevent the formation of biofilms. In order to determine whether the experimental materials have anti-biofilm effects, we first observed the biofilms of Staphylococcus aureus and E. coli using SEM, and then used ConA-FITC staining to further observe the differences in biofilms between the groups. After the differences were observed, the CV staining method was used to quantify the biofilms in each group. The biofilm test results showed that GO and ε-PLL coating significantly inhibited the biofilm formation ability of these two bacteria.
Cellular lactase can deacetylate 2ʹ,7ʹ-2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) into non-fluorescein DCFH, which can be oxidized by intracellular ROS to fluorescein 2ʹ, 7ʹ-Dichlorofluorescein (DCF). Therefore, the fluorescence intensity of DCF can reflect the level of ROS to a certain extent. 46 In this study, the ROS level of the experimental group increased significantly, indicating that ROS level is one of the main antibacterial mechanisms of GO and ε-PLL.
According to appearance and morphology, perforation was more common in GO group, and atrophy was more common in ε-PLL group. We have previously conducted a detailed review of the antibacterial mechanism of GO37 on the surface of the material. In addition to oxidative stress, GO reduces microbial activity through physical damage, while ε-PLL destroys bacteria mainly through electrostatic action, affecting cell membrane permeability and causing cell disintegration (Figure 20). 41,47,48 Whether the hydrophilicity and hydrophobicity of the surface is one of the mechanisms that affect the surface antibacterial activity has not been determined. The results of this study show that the antibacterial activity of the surface of the material has nothing to do with the hydrophilicity and hydrophobicity of the surface. Figure 20 Antibacterial and biofilm resistance mechanism of GO and ε-PLL on CoCrMo alloy.
Figure 20 Antibacterial and biofilm resistance mechanism of GO and ε-PLL on CoCrMo alloy.
Coating materials can generally be divided into two categories: one is the combination of antibiotics and polymers, and the other is the type that relies on the intrinsic properties of the material to kill or repel bacteria on the surface. 15 The antibacterial properties of the material surface can generally be divided into four categories: anti-sticking surface, contact killing/bacteriostatic surface, release killing/bacteriostatic surface, and remote control sterilizing surface. In this study, we found that after coating, the adhesion rate of bacteria on the surface of the material is reduced, and a large number of bacteria attached to it die. In the bacteriostatic zone test, we did not find the existence of bacteriostatic zone. Therefore, the experimental group did not release something to kill the bacteria. Therefore, in terms of classification, the coating in this study is an inherent property of the material, which can kill or repel bacteria, has anti-adhesive properties and contact kill/inhibit surfaces.
However, no matter what kind of coating, its adhesion is a problem that cannot be ignored. The elastic modulus of each group was measured by nanoindentation. In the experimental group containing GO, both the elastic modulus and Vickers hardness decreased, which may be because GO is not a single-layer structure and the interlayer bonding is not strong. The application of ε-PLL solves this problem well. Compared with the control group, there was no significant difference in the elastic modulus and Vickers hardness of the group containing ε-PLL. Here, ε-PLL can act as an adhesive to fix itself and GO to the alloy surface. However, not all antimicrobial coatings need to adhere firmly to the surface of the substrate.
Taking the artificial joint prosthesis as an example, it is well known that the interface of the artificial joint prosthesis is generally divided into a movable interface and a fixed interface. In general, infection mainly occurs on the fixed interface, while wear mainly occurs on the moving interface. In the case of artificial knee joint prostheses, the fixed interface, especially the interface between the polyethylene pad and the tibial plateau, is one of the most common sites for bacterial growth. Therefore, eliminating the infection of the fixed interface is equivalent to solving the infection of the artificial joint prosthesis to a large extent. In this study, we successfully prepared the coating and proved that the coating has antibacterial and anti-biofilm effects. However, for the further optimization of the coating process, there is still a long way to go before clinical application.
Through exploration and optimization of experimental parameters, GO and ε-PLL coatings were successfully prepared on CoCrMo alloy by electrodeposition. The results show that the coating not only has good mechanical properties, but also has obvious antibacterial and anti-biofilm effects on Staphylococcus aureus and Escherichia coli in vitro. In the era of increasingly serious prosthesis-related infections, coating materials provide a promising new method for solving the problem of orthopedics and dental implant infections.
This work was supported by the National Natural Science Foundation of China (82072402 and 81672189) and the Natural Science Foundation of Shaanxi Province (2020JQ-965).
The authors report no conflicts of interest in this work.
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