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Back to Journal »International Journal of Nanomedicine» Volume 14

Research on Formation of Silver Nanoparticles on Titanium Surface by Ion Implantation Technology

Author Lampé I, Beke D, Biri S, Csarnovics I, Csik A, Dombrádi Z, Hajdu P, Hegedűs V, Rácz R, Varga I, Hegedűs C 

Published on July 1, 2019, Volume 2019: 14 pages 4709-4721

DOI https://doi.org/10.2147/IJN.S197782

Single anonymous peer review

Editor approved for publication: Prof. Dr. Anderson Oliveira Lobo

István Lampé, 1 Dezső Beke, 2 Sándor Biri, 3 István Csarnovics, 4 Attila Csik, 3 Zsuzsanna Dombrádi, 5 Péter Hajdu, 3 Viktória Hegedűs, 6 Richárd Rácz, 3 István Vargeda, University, 7 Csaba, Debrecen, Hungary; 2 Department of Solid State Physics, University of Debrecen, Hungary; 3 Hungarian Academy of Sciences, Institute of Nuclear Research, Debrecen, Hungary; 4 Department of Experimental Physics, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary Sen; 5 Department of Medical Microbiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 6 Department of Orthodontics, Faculty of Dentistry, University of Debrecen, Debrecen, Hungary; 7 Department of Periodontology, Faculty of Dentistry , University of Debrecen, Debrecen, Hungary Purpose: The use of titanium implants has become a widely accepted and used method of replacing missing dentition. Obviously, inflammation around the implant will occur in many cases. The purpose is to create and evaluate the antibacterial effect of silver nanoparticles (Ag-NP) coated titanium surfaces, if applied to the surface of dental implants, can help prevent such processes. Method: Use annealing I, electron cyclotron resonance ion source (ECRIS) beam injection of silver ions, silver physical vapor deposition (PVD), annealing II procedures to create a securely anchored Ag-NP layer on 1x1 cm2, and Ti sample 2 . The antibacterial effect was evaluated by culturing Staphylococcus aureus (ATCC 29213) on the surface of the sample for 8 hours, and comparing the results with the results of the glass and pure titanium samples as controls. Alamar Blue assay was performed to check cytotoxicity. Result: It is proved that silver nanoparticles are present on the treated surface. The particles have an average diameter of 58 nm, a deviation of 25 nm, a Gaussian distribution, and a fill factor of 25%. The antibacterial evaluation showed that the sample covered with nanoparticles had an antibacterial effect of 64.6%, which was statistically significant. The test also proved that the nanoparticles are securely fixed on the titanium surface and have no cytotoxicity. Conclusion: Creating a layer of silver nanoparticles can be an option to add antibacterial functions to the surface of the implant and help prevent the inflammatory process around the implant. Recent studies have shown that silver nanoparticles can induce pathological changes in mammalian cells, so the safe fixation of the particles is essential to prevent them from entering the circulation. Keywords: implant, inflammation around implant, oxide layer, cell compatibility, antibacterial effect, physical vapor deposition, geometric model

In the past few decades, the use of titanium dental implants to restore lost dentition has become a widely accepted routine in daily dental practice. With the significant increase in the number of positioned implants, the awareness of certain risks that affect the life of the prosthesis has become the focus of attention. The inflammatory process of the soft and hard tissues around the implant can lead to chronic inflammation, leading to the loss of the implant and the loss of the entire restoration. Based on a meta-analysis, Derks and Tomasi1 estimated the weighted average prevalence of peri-implant mucositis and peri-implantitis to be 43% and 22%, respectively. This statistically high-level inflammatory process has led to the development of different non-surgical and surgical treatment procedures, but the results are questionable. This high level of inflammation turns attention to preventing the process. In addition to strict adherence to oral hygiene and follow-up procedures guidelines, if the system itself has an inherent anti-inflammatory effect, it will be beneficial to be constantly vigilant to prevent and fight inflammation. Silver has a well-known antibacterial effect and is toxic to bacteria. The antibacterial effect of silver ions is based on many factors, such as inactivating enzymes, binding and denaturing DNA, and interfering with cell membrane transport. Recently, several authors have used different methods to study the antibacterial effect of silver nanoparticles of different sizes and shapes layered on the titanium surface, but the effect is still unclear. Wang et al. 2 demonstrated the extracellular and intracellular reactive oxygen species of bacteria placed on a titanium surface covered by silver nanoparticles. The explanation for the development of reactive species is believed to be the microcurrent effect between silver and titanium.

Many chemical and physical methods are known for silver modification of titanium/titanium oxide surfaces. 3-7

In most applications, the size distribution, density, adhesion, and depth distribution of silver nanoparticles on the surface are important because these parameters determine mechanical stability, antibacterial activity, optical properties, and photosensitivity. Their efficiency in charge carrier generation, plasma field distribution, and possible effects on biological activity depends on their size and other geometric parameters. In the case of the combination of physical vapor deposition (PVD) and electron cyclotron resonance (ECR), the geometric parameters of the particles can be controlled by the initial layer thickness, injection parameters, heat treatment time and temperature, just as before for gold nanoparticles ongoing. 8

This article focuses on a new technology for producing silver nanoparticles with a defined size and distribution on the titanium surface, and the impact of these nanostructures on bacteria. The surface of the titanium sample is affected by the complex physical pathways of Ag-NP formation: thin Ag layer deposition, ion implantation and thermal annealing. The antibacterial properties of the surface were studied against Staphylococcus aureus. The influence of the technology and the resulting nanostructure parameters, their influence on the surface structure, and the influence on the antibacterial process are studied.

The results of Shi et al., 9 Pacurari et al., 10 Rahman et al., 11 and Doudi and Setorki12 showed that nanoparticles, including circulating silver nanoparticles, can cause pathology, such as atherosclerosis, DNA damage, and neurotoxicity And liver toxicity. Inkielewicz-Stepniak et al. 13 demonstrated that when silver nanoparticles are present, the cytotoxicity of fluoride to fibroblasts increases.

These data indicate the importance of fixing silver nanoparticles on the titanium surface to prevent them from entering the circulation. The purpose of this study is to evaluate a new method for producing repairing nanoparticles and its antibacterial effect.

We hypothesize that the Ag-Np Ti surface created by this new ion implantation technique has a significant antibacterial effect.

In order to achieve the previously described titanium surface covered by anchored silver nanoparticles, the original titanium surface was modified in four stages: Annealing I The beam implantation of silver ions by electron cyclotron resonance ion source (ECRIS) silver physical vapor deposition (PVD) Annealing II

The resulting surface is analyzed by physical analysis research methods. These analysis methods are as follows:

Previously, the simulation of silver ion implantation was implemented by SRIM computer code to estimate the penetration depth of the ion to be implanted. 14 SRIM is a software compilation that can estimate many parameters of ion transport in substances. In this work, in order to calculate the depth of the stop ion, the ion stop and range application in the target is used, which can also manage complex multi-layer configurations. SRIM also includes a quick calculation option to create ion blocking distributions and even blocking power meters. These additional functions help to obtain more information about the silver ions that stop entering the titanium dioxide layer.

SNMS is a unique thin film analysis method that uses post-ionization technology. In this method, neutral atoms are sputtered from the surface of the sample with the help of plasma such as Ar, Kr, and Ne, and are detected after post-ionization. For sputtering and post-ionization processes, it is used as an inductively coupled low-voltage radio frequency discharge. It is a suitable technique for measuring the chemical composition of almost any type of sample, because the atomic flux sputtered from the sample represents the topmost stoichiometry.

In order to evaluate the thickness of the TiO2 layer before and after annealing, an I sputtering experiment was carried out in the INA-X type SNMS system produced by SPECS GmbH in Berlin. 15 INA-X equipment uses electron cyclotron wave resonance (ECWR) plasma as the ion source for sputtering and post-ionization, with a sputtering energy of 100 eV. SNMS is equipped with a quadrupole mass spectrometer (Balzers QMA 410) up to 340 AMU for element detection. The bombardment energy of Ar+ ions on the sample is 350 eV, the plasma pressure is 1.5 mbar, and the current density is ~1 mA/cm2. A circular sample area with a diameter of 2 mm was sputtered through a Ta mask. By using a profiler to calibrate the sputtering rate, the sputtering time is converted to depth.

The surface roughness is measured by the Ambios XP-I profiler (Ambios Technology, USA) in online scanning mode. Linear scanning stage and high-precision optical plane measure nanometers on time. The horizontal resolution is limited to a maximum of 60,000 data points per scan line. By measuring the vertical stylus displacement as a function of position, we can measure the surface roughness with nanometer vertical resolution with a stylus load of 1 mg.

The morphology of the radiated and sputtered surface was studied by SEM (Hitachi S4300-CFE, Japan). This is an electron microscope that produces an image of the sample by scanning the surface with a focused electron beam. Electrons interact with atoms on the surface to generate various signals, which contain information about the topography and composition of the surface. This technology can capture high-resolution images with high depth of field. In order to obtain spatially resolved information about the formed particles, SEM images were taken. The nanoparticles are clearly visible on the titanium surface. These nanostructures are evaluated by a computer program based on National Instruments' Vision Assistant 2011. After the microbiological evaluation, the samples processed by the ultrasonic equipment are again inspected by SEM to check whether there are nanoparticles even after the ultrasonic treatment. In addition to the morphological characteristics, several other characteristics of the test sample can be tested according to the installed equipment (such as element composition).

The composition of the surface was studied by an energy dispersive spectrometer (Bruker XFlash 6, Germany, installed in the SEM unit). EDS is an analytical technique used to define the elemental analysis of substances. Since each element has a unique atomic structure, there is a unique set of peaks in the electromagnetic emission spectrum. In order to induce the characteristic X-ray emission of the sample, an accelerating voltage of 15 keV was applied during the measurement. The X-ray spectrum is detected by a silicon drift detector (SDD).

Use a grade 2 titanium plate (99.6 at%, grade 2, Spemet Co., Taipei, Taiwan) with a size of 10x10 mm. A Struers LaboPol-35 polisher equipped with Struers LaboForce-Mi equipment (Struers, Denmark) was used to mechanically polish the plate to #2000 grit, and then 3 µm diamond was used to produce a mirror-like surface. Then the substrate was cleaned 3 times in acetone for 10 minutes each time. Place them in an ultrasonic cleaning tank at room temperature, rinse with distilled water for 30 minutes, and then air dry.

This titanium plate was used as a sample of Gr.1. These plates are used in the further stages of surface treatment. For Gr.2, silver nanoparticles are produced by annealing I, ECRIS, PVD and annealing II procedures, as follows.

One of the novelties of this work is the technique of preparing silver nanoparticles on the surface of titanium. This technology has been used to create silver and gold nanostructures on different substrates. 8,16

The thickness of the oxide layer on the prepared surface was measured by SNMS. In order to increase the thickness of the oxide layer produced by Ag-NP, annealing was carried out. All samples were annealed at 550°C for 5 hours under normal atmospheric conditions.

14.3 GHz electron cyclotron resonance ion source (ECRIS) can be used from H, He, CO, CH4, N, O, Si, Ne, Ar, Kr and Xe gases and C, C60, Ca, Fe, Ni, Zn, solids Ag and Au ions. 17,18 In this study, a silver ion beam production method was developed. Neutral silver atoms are evaporated in a commercial filament furnace (Pantechnik, France). The furnace head is placed 4 cm away from the injection plate in the plasma chamber and filled with 170 mg pure silver (99.999%). The best working temperature is between 850 and 950°C. Oxygen is used as a supporting gas. The composition of the beam is: Ag: 3.8%, O: 18%, and others (H, C, OH, H2O): 68%. A typical silver ion beam spectrum is shown in Figure 1. Figure 1 Silver ion beam spectrum (optimized for Ag1+).

Figure 1 Silver ion beam spectrum (optimized for Ag1+).

The plasma is optimized for Ag.1+. For the charge state (Q=1–4), a silver beam current between 0.02 and 0.5 eµA is obtained (Figure 2), and the average charge state qav=1.4. The size of the ion beam is a circle with a diameter of 50 mm. The ion beam from the plasma is extracted at a voltage of 2 kV. In our setup, all the extracted plasma components reach and hit the sample. The implantation depths of charged ions from 1 to 4 are different. The Ti sample was injected at a dose of 1.5*1016ion/cm2 Ag. Figure 2 SEM images and EDS spectra of the surface before (A and B) and after (C and D) ECR+PVD treatment. The signal from the silver nanoparticles is detected. Abbreviations: ECR, electron cyclotron resonance; EDS, energy dispersive X-ray spectroscopy; PVD, physical vapor deposition; SEM, scanning electron microscope.

Figure 2 SEM images and EDS spectra of the surface before (A and B) and after (C and D) ECR+PVD treatment. The signal from the silver nanoparticles is detected. Abbreviations: ECR, electron cyclotron resonance; EDS, energy dispersive X-ray spectroscopy; PVD, physical vapor deposition; SEM, scanning electron microscope.

After ion implantation, a 7-8 nm thick silver layer was deposited on the titanium sample. A thin layer is deposited from a pure silver target by magnetron sputtering technology. The working gas is Ar, and the deposition is carried out for 12 seconds at a current of 6 mA. Therefore, when the substrate is at room temperature, the deposition rate is 0.6 nm/s.

The sample was annealed in Ar:H atmosphere at 550°C for 15 minutes to form Ag-NP. Since the TiO2 layer is quite thin, due to the heat treatment, the Ti-Ag phase can be formed without forming nanoparticles. To solve this problem, a thicker TiO2 layer was produced by thermal annealing (annealing I). The thickness of the TiO2 layer after annealing was measured by SNMS. After annealing, a thin silver layer and silver nanoparticles are formed in the same manner as described above.

According to Wang et al., the Gram-positive control strain Staphylococcus aureus (ATCC 29213) was used to test the antibacterial activity of each group of samples. 2 The bacterium was subcultured twice overnight on Columbia blood agar at 37°C. Prepare a bacterial solution with a density of 0.5 McFarland in brain heart infusion broth (BHI) and dilute to a concentration of approximately 1×106 colony forming units (CFU)/mL.

The experiment was carried out in accordance with the ISO22196:2007(E) protocol. 19 Place a 1 cm2 sample on a glass microscope slide in a petri dish; pipette 50 µL (0.05 mL/cm2) of bacterial suspension onto the surface of each sample, and then carefully cover the glass cover with a sterile borosilicate microscope To prevent leakage of the suspension at the edge. Then incubate 20 petri dishes in the dark at 37°C.

After 8 hours of incubation, place the sample with a cover glass in 1 mL of BHI broth, mix by vortexing vigorously for 5 minutes, and then sonicate it for 3 minutes to separate the bacterial cells from the surface. 7,21 Serial dilution in BHI broth was used for colony counting by Herigstad et al.'s drop plate (DP) method, 22 and 50 µL of each suspension was distributed on Mueller-Hinton agar at five evenly spaced 10 µL droplets. After dripping dry, turn the petri dish upside down and incubate at 37°C for 1 night, and record the total number of CFU per culture plate in the diluent that can be counted. The cultivation is carried out in complete darkness to prevent photocatalysis on the titanium surface.

The detachment efficacy was determined on a control test piece from each sample group, which was inoculated with a bacterial suspension and detached immediately without any incubation (zero time control). Each test is repeated, and each group of experiments is repeated at least 3 times. The antibacterial rate is determined by the following formula: Antibacterial rate = (1-(CFU experimental group/CFU control group)) x100%.2

A microscope slide (10x10 mm size) is used as the control surface.

The samples are used to evaluate the antibacterial effect after standard sterilization procedures in an autoclave.

The antibacterial rate data of the two surfaces (titanium and silver-plated titanium) are compared with the value 0 corresponding to the glass control surface by a single-sample Student's t-test, while the independent sample t-test is used to compare the two surface data. In addition, the margin of error of the average estimate is also calculated. Please note that for the above method, it is necessary to assume the normality of the antibacterial data (because otherwise only a relatively large sample can be used for testing).

Alamar Blue assay® (Thermo Scientific, Waltham, MA, USA) was used to detect cell viability on the surface of different materials (glass, Ti and Ag-Np implanted Ti) to detect dental pulp stem cells (DPSC). 4×104 cells were seeded on the surface of the research material, and then cultured in DMEM F12 (Gibco, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% Glutamax (Gibco, Waltham, MA, USA) for 14 days (Gibco) and 1% antibiotic-antifungal agent (Gibco). The medium is updated 3 times a week. Finally, perform the Alamar Blue measurement according to the manufacturer's instructions. In short, replace the cell culture medium in each well with 10% (v/v) Alamar Blue solution in each well, and after incubating for 1 hour at 37°C, incubate in a humidified atmosphere containing 5% CO2 After 4 hours at 37°C, the fluorescence of the sample (100 µL) was measured using a microplate reader (HIDEX Sense Turku, Finland) under 544 nm excitation/595 nm emission.

The DPSC cells on the surface of the material were evaluated by SEM. After the samples were fixed with 2% (v/v) glutaraldehyde for 2 hours and 1% OsO4 for 1 hour, graded ethanol solutions (10, 30, 50, 70, 80, 90 and 100% (v/v), 15 minutes each) and use CO2 to dry the critical point. Finally, they were sputtered gold-plated for 30 seconds. The plasma current is 18-20 mA, and the sputtering Ar pressure during the coating process is 10-20 MPa. A scanning electron microscope (SEM, Hitachi S4300 CFE, Tokyo, Japan) was used to image the sample at an accelerating voltage of 10 kV.

The oxide layer on the titanium surface increased after annealing I, and was studied by SNMS. However, the roughness of the titanium surface will affect the results of the SNMS measurement. In our example, the average surface roughness is as high as Ra=8 nm, which means there are several peaks and valleys on the nanometer scale. Therefore, no sharp curves are observed on the SNMS depth profile, so the thickness of the oxide layer can only be estimated: about 25 nm.

SEM has been used to obtain 2D information of the surface configuration. Obvious evidence was observed that nanoparticles were formed on the titanium surface due to annihilation after ion implantation and physical vapor deposition. Due to the nanoparticles, the surface morphology has changed drastically (Figure 2A and C). Due to the influence of processing, the average roughness of the sample increased from Ra=8 nm (Rmax=76 nm) to Ra=62 nm (Rmax=113 nm). The elemental analysis of the surface by the EDS built into the SEM proves that the structure formed on the titanium surface is silver nanoparticles (Figure 2D).

The SEM image is evaluated by a computer program based on National Instruments Vision Assistance. The results show that the nanoparticles cover the surface with a fill factor of 25%. The diameter distribution of the nanoparticles follows the Gaussian trend shown in Figure 3. The median is 58 nm and the deviation is 25 nm. The titanium peak is much higher than the silver peak because the interaction volume of EDS is on the order of micrometers, while the size of silver nanoparticles is on the order of nanometers. Figure 3 Diameter distribution density of silver nanoparticles (SNPs). Abbreviations: ECR, Electron Cyclotron Resonance; PVD, physical vapor deposition.

Figure 3 Diameter distribution density of silver nanoparticles (SNPs). Abbreviations: ECR, Electron Cyclotron Resonance; PVD, physical vapor deposition.

In order to check the physical stability of Ag-NP on the titanium surface of the sample used for antibacterial evaluation, ultrasonic treatment was carried out and analyzed by SEM. Compare the SEM image with a sample with AG-NP without ECRIS. (In this case, Ag-NP-s was produced by annealing I, PVD and annealing II procedures.) As can be seen in Figure 4C and D, when the PVD method is applied, the ultrasonic treatment releases from the surface As long as silver nanoparticles. A detailed SEM study shows that the peeling of the oxide layer is the reason for the removal of Ag particles. In Figure 4E, the boundary between the free surface (retained after exfoliation) and the surface still covered by the remaining oxide layer with Ag nanoparticles is shown. The EDS spectrum shows that the oxygen content of the above two regions is quite different (Figure 4F). The fill factor has changed from ~25% to ~1‰, as shown in Figure 4B and D. For Gr.2 (ECR+PVD) samples, ultrasonic treatment proved that the particles on the treated surface have high physical stability (see Figure 4A and B). However, several small areas (see arrows) in Figure 4B show that the oxide layer and Ag particles have similar peeling, but the relative area of ​​these areas is much smaller than the sample without ECR treatment. In this case, the distribution has changed within the error range. One of the possible reasons is that the injected silver ions are fixed on the surface by the ECR ion source treatment. It is worth noting that compared with normal tissue conditions, the physical stimulation of ultrasound therapy is much greater. Figure 4 ECR+PVD surface is compared with the sample whose surface is modified only by PVD ​​, and the proof of the effect of ultrasonic treatment on surface nanoparticles. (C and D) When only the PVD method is applied, ultrasonic treatment releases nanoparticles from the surface. (A and B) ECR+PVD changes only slightly (arrow). (E) A detailed investigation shows that the peeling of the oxide layer is the reason for the removal of Ag-Np-s. The ECR+PVD method also peeled off (arrow on b), but this area is significantly lower than the area where only PVD is used.​​ (F) The oxygen content of the peeled and unpeeled areas is significantly different, indicating that the TiO2 layer has been removed. Abbreviations: ECR, electron cyclotron resonance; PVD, physical vapor deposition.

Figure 4 ECR+PVD surface is compared with the sample whose surface is modified only by PVD ​​, and the proof of the effect of ultrasonic treatment on surface nanoparticles. (C and D) When only the PVD method is applied, ultrasonic treatment releases nanoparticles from the surface. (A and B) ECR+PVD changes only slightly (arrow). (E) A detailed investigation shows that the peeling of the oxide layer is the reason for the removal of Ag-Np-s. The ECR+PVD method also peeled off (arrow on b), but this area is significantly lower than the area where only PVD is used.​​ (F) The oxygen content of the peeled and unpeeled areas is significantly different, indicating that the TiO2 layer has been removed. Abbreviations: ECR, electron cyclotron resonance; PVD, physical vapor deposition.

Staphylococcus aureus (a common gram-positive bacteria) was used to evaluate the antibacterial properties of the prepared surface. After culturing the bacterial cells on each sample for 8 hours, the bacteria were collected according to the DP method and re-cultured for colony counting. Figure 5 shows the antibacterial rate (%) of each test sample. Compared with the inert control glass surface, the Gr.1 sample actually has no effect on bacterial growth (P value> 0.8, and the margin of error in the average estimate is also quite large, 27.1%). In the case of a surface containing Ag-NP, the inhibitory effect on bacterial growth is significantly stronger. Our results show that the antibacterial effect is reproducible, and the inhibitory effect is significant compared with the inert glass control and the ineffective Ti surface. The average antibacterial rate of the Gr.2 sample is 64.6%, the margin of error is 19.6% (the confidence level is 90%), and the average standard error is 6.7%. The effect in Ag-NP is statistically significant. The sample is comparable to the control glass surface. Ratio (P value = 0.010), and also significant compared to Ti surface (P value = 0.005). The bars in Figure 5 show the antibacterial rates of titanium and silver-plated titanium surfaces with standard deviations compared to the control glass surfaces. Each column shows the average of three independent measurements in each case and the margin of error (mean ± margin of error) at the confidence level of 90%.

The bars in Figure 5 show the antibacterial rates of titanium and silver-plated titanium surfaces with standard deviations compared to the control glass surfaces. Each column shows the average of three independent measurements in each case and the margin of error (mean ± margin of error) at the confidence level of 90%.

Although the antibacterial properties of implant materials are a very useful phenomenon, the materials and/or coating strategies used should not have a cytotoxic effect, especially because they will be used for a long time. The cell viability on the surface of different materials is shown in Figure 6. As a control, cells were seeded on a glass surface, and an untreated titanium surface was used to test the cytocompatibility of the implanted silver nanoparticles. After 14 days, compared with the glass surface control, the untreated Ti and Ag-Np-infused Ti surfaces both showed unchanged cell viability. These results indicate that the implanted Ag-Np has good long-term use biocompatibility, which has good characteristics when used in dentistry or other medical fields. Figure 7 shows the SEM image of dental pulp stem cells (DPSC) on the Ti surface implanted with Ag-Np. Figure 6 Alamar Blue determination of cell viability on different surfaces (glass, untreated Ti, Ag-Np implanted Ti) on 14 days (n=3). Figure 7 SEM image of Ag-Np implanted DPSC on Ti surface. Abbreviations: DPSC, dental pulp stem cells; SEM, scanning electron microscope.

Figure 6 Cell viability measurement of Alamar Blue on different surfaces (glass, untreated titanium, Ag-Np implanted titanium) for 14 days (n=3).

Figure 7 SEM image of Ag-Np implanted DPSC on Ti surface. Abbreviations: DPSC, dental pulp stem cells; SEM, scanning electron microscope.

The use of osseointegrated dental implants to restore lost dentition has become a widely accepted and routinely used method in modern dental restorations. With the rapid increase in the number of implants implanted in the early 1990s, certain complications have appeared and there is a threat of losing previously implanted implants. Lindhe and Meyle23 presented the data of their working group at the European Symposium on Periodontology in 2008, and found inflammation around the implant in many dental implant patients. More than 50% of implant patients find peri-implant mucositis, and the proportion of peri-implant inflammation is between 28% and 56%. There may be several factors behind it, such as improperly designed or manufactured restorations, overload, poor oral hygiene or poor hygiene, or surgical errors during placement. Systemic diseases or medications can increase the risk of inflammation around the implant. According to the guidelines of the European Association of Dental Implantologists at the 10th European Implant Consensus Conference (EuCC) held in Cologne in 2015, inflammation around implants can be divided into three forms: 24 Mucositis is the initial form and is reversible , And showed signs of gum inflammation. -Implantitis is a more serious disease. Patients with periapical inflammation or nonhealing of periapical lesions due to bone burns during the operation will develop retrograde peri-implantitis with bone loss.

In the process of treatment, risk factors must be eliminated, while bacteria must be eliminated to rebuild a healthy environment. Treatment includes the application of antibiotics and antibacterial agents, augmentation and other surgical techniques. As with other diseases, prevention should be given top priority to prevent the development of pathology. Due to the high incidence of inflammation around the implant, it would be advantageous if the implant itself has antibacterial properties.

For thousands of years, silver has been known for its antibacterial effects. As far as its effects are concerned, its application in community health projects and personal antibacterial treatment procedures is becoming more and more widespread. Although the exact path of this effect remains to be studied, it is agreed that it has multiple points of attack. Silver ions change the function of bacterial cell walls and cell membranes. The combination of silver ions and sulfur can block the functions of proteins and enzymes containing sulfate groups. Silver ions bind to DNA and prevent the replication process. 25 Zheng et al. 21 discovered the gene expression of glucan binding protein B in Str. Proteus, Porphyromonas gingivalis fimbrin A and lectin-like sequence 4 in Candida albicans were significantly reduced on the Ag-NP surface, confirming its antibacterial effect. Glucan binding protein B plays an important role in cell wall construction, while fimbrin A in Porphyromonas gingivalis and lectin-like sequence 4 in Candida albicans play a key role in adhesion. Reducing the expression of these genes will produce antibacterial effects.

Klueh et al. 26 proposed several antibacterial mechanisms of silver ions:-React with enzymes containing sulfhydryl groups in the cell wall to change transmembrane energy and electrolyte transport mechanisms-Block cytochrome oxidase and NADH-succinate dehydrogenase to change respiration Mechanism Cell-binds to bacterial DNA and causes denaturation by changing the bond between purine and pyrimidine

Zare and Shabani27 described phenomena that may lead to bacterial death:-Interruption of ATP production and DNA replication by ingestion of Ag ions-Interaction of Ag ions with bacterial enzymes to interfere with bacterial protein synthesis-Interaction of Ag with bacterial enzymes directly damages the cell membrane peptidoglycan wall and Plasma membrane

Akhavan and Ghaderi28 found that direct contact between bacteria and sharp nanoparticles can cause mechanical changes and destruction of bacterial walls and cell membranes.

Wang et al. 2 proved that Ag-NP has an antibacterial effect on Ti complex due to the electron transfer caused by the microcurrent effect between Ti and Ag-Np-s and the induction of reactive oxygen species in the cell.

Prakash et al. 29 reported that the synergistic effect of the nanocomposite layer of Ag and TiO2 on the antibacterial activity is stronger than the antibacterial effect of Ag and TiO2 alone.

According to Liu et al.,30 the mechanism of this synergistic effect is that TiO2 promotes the release of Ag ions and increases the surface energy.

Mao et al. [31] mentioned that the penetration of silver and zinc ions into bacterial cells would damage the bacterial wall and intracellular structure. Jin et al. 32 and Mao et al. 31 also mentioned that silver ions stimulate the immune system by producing a large number of white blood cells and neutrophils.

Silver used in the form of nanoparticles has a significantly higher surface, which contributes to its more effective antibacterial effect. On the other hand, nanoparticles are too small to be cleared by the immune system, and they may cause toxicity or inflammation. 33 Several studies have shown that silver nanoparticles are cytotoxic to several cell lines. 34 Shi et al. 9 proved that silver nanoparticles may cause damage and dysfunction of human endothelial cells, thereby causing harm to atherosclerosis. Inkielewicz-Stepniak et al.13 found that exposure of human gingival fibroblasts to Ag-Np-s causes increased cell damage, which affects cell membranes and mitochondria. Kang et al.35 demonstrated that Ag-NP-s in cells can induce cell death by inducing the production of reactive oxygen species, DNA damage, or damage to mitochondrial function. The paper shows that Ag-NP-s induces cell apoptosis by generating ROS. 36

There are many ways to produce silver nanoparticles on the titanium surface. The technique used in the current experiment ensures the stable anchoring of Ag nanoparticles on the titanium surface. The ECRIS technology was used to implant silver ions and then annealed to produce a stable surface configuration of silver nanoparticles with an average diameter of 58 nm and a fill factor of 25%. In the current study, the colonization of Staphylococcus aureus on the surface was investigated. The results show that, compared with the natural titanium surface or the control glass surface, the resulting surface has a significant antibacterial effect on Staphylococcus aureus. We have demonstrated that to reach the highest column in Figure 5, all four steps of the combined treatment are required to have physically stable Ag-NP-s on the antibacterial surface.

In our experiments, we managed to create a titanium surface covered by silver nanoparticles, and it has a significant antibacterial effect. The diameter distribution of nanoparticles follows a Gaussian trend; the median value is 58 nm, and the deviation is 25 nm. The filling effect is 25%. Figure 2 (left) shows an SEM image of the surface with anchored nanoparticles, while Figure 3 shows the associated Gaussian curve.

Cao et al. 37 reported that Ag-NP-s with a larger size (5-25 nm in diameter) have better antibacterial effects than Ag-NP-s with a smaller size (4 nm in diameter). The technology used ensures that the nanoparticles are securely anchored to the titanium surface, and their role in the antibacterial effect exhibited is irrelevant. Only the released Ag ions can produce this effect.

Since the antibacterial effect of silver is known, we believe that contact of the larger surface of silver with bacteria will enhance the antibacterial effect of the surface. One can explain the role of silver nanoparticles by adding an antibacterial surface. In order to understand what is the optimal geometric arrangement of Ag nanoparticles, the following geometric considerations were made.

Our geometric model treats nanoparticles as hemispheres; the surface area and volume are calculated as follows:

The thickness d of the deposited continuous silver film is given by (1)

Where VAgo is the volume of the film, and Ah is the surface of the substrate covered. The effective surface of the bead film is given by (2)

Where N is the number of Ag particles and Ao is the surface of a (spherical) particle: (3)

Assuming that all particles have the same radius ro, the volume of silver in the beads is (4​​)

Where Vo is the volume of a (hemispherical) particle: (5)

If a certain amount of Ag is lost in the process of forming a beaded film (for example, by evaporation, diffusion into the substrate, falling off from the particles, etc.), then (6)

The relationship α<1 holds.

Using equations (1), (4) and (5), we can get: (7)

The number of particles is (8)

Substituting it into equation (2), the effective antibacterial surface (silver particle surface) is (9)

Or its relative value (10)

The measurable relative coverage in SEM photos is only two times different from this (the surface of a hemispherical particle is twice the area ro of a circle with the same radius) (11)

Now, using the numbers obtained in the experimental part: d≅8 nm, ro=29 nm, and ηSEM≅0.25, we get (12)

It should be noted that the d/ro ratio is a key factor for optimizing the antibacterial effect and cannot be controlled by simple geometric operations. It is well known that the particle radius (ie the value of η) formed in the beading process of a film with a thickness of d depends on the temperature and time of the heat treatment, the initial grain size of the film, and evaporation. 38 In addition, the shedding factor α also depends on the experimental conditions and is also related to the adhesion strength. Therefore, further improving the resistance to ultrasound therapy and the increase in relative coverage are challenges for further research.

The investigation proved our hypothesis that the technology used in this study can produce titanium surfaces with anchored silver nanoparticles, which has a significant antibacterial effect. If produced on the surface of dental implants, it can help prevent the development of peri-implant inflammation. . Further studies are planned to examine the cytotoxicity of the nanoparticle layer and optimize the surface configuration to achieve higher antibacterial rates if possible. It can also achieve a higher antibacterial rate by using a smaller amount of silver to increase the η value, thereby producing an antibacterial surface in a more cost-effective manner.

Ag-Np, silver nanoparticles; ECRIS, electron cyclotron resonance ion source; PVD, physical vapor deposition; ECR, electron cyclotron resonance; SRIM, stop and ion range in matter; SNMS, secondary neutral mass spectrum; EDS, energy dispersion X-ray spectroscopy; CFU, colony forming unit; BHI, brain heart infusion; ROS, reactive oxygen species; DPSC, dental pulp stem cells.

The collection of human dental pulp tissue in this study was approved by the Ethics Committee of the Hungarian Medical Research Council (approval number: ETT TUKEB 49849-3/2016/EKU). For the use of tissue samples, written information consent has been obtained from all human subjects participating in the survey.

The research was funded by the Excellence Program of Higher Education Institutions of the Ministry of Human Resources of Hungary, within the framework of the Biotechnology Project of the University of Debrecen, and received GINOP-2.3.2-15-2016-00011 and GINOP-2.3.2-15- 2016-00022 project. These projects are jointly funded by the European Union and the European Regional Development Fund.

The authors report no conflicts of interest in this work.

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