In vitro biocompatibility of anodized titanium with deposited silver nanodendrites
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Engineers searching new dental biomaterials try to modify the structure of the material in order to achieve the best performance as well as increased migration and proliferation of cells involved in the osseointegration of the implant. In this work we show in vitro test results of the Ti, which was anodically oxidized at high voltages with additionally deposited silver in the form of nanodendrites. The in vitro cytocompatibility of these materials was evaluated and compared with a conventional microcrystalline titanium. During the studies, established cell line of human gingival fibroblasts (HGF) and osteoblasts were cultured in the presence of tested materials, and its survival rate and proliferation activity were examined. Titanium samples modified with silver has a higher degree of biocompatibility in comparison with the unmodified reference material. Cells in contact with studied material showed a higher relative viability potential, stable level of proliferation activity, and lower rate of mortality. Biocompatibility tests carried out indicate that the anodically oxidized titanium at high voltages with additionally deposited nanosilver could be a possible candidate for dental implants and other medicinal applications.
Titanium (Ti) and its alloys are most useful and most often investigated metallic biomaterials. Titanium possesses high strength to density ratio, relatively low Young modulus value, very good corrosion resistance, and biocompatibility. For providing fast osseointegration and long-term usage in the human body, the implant surface should be modified, i.e., it should be rough or porous, oxidized, and covered by biocompatible coating including calcium–phosphate compounds [1, 2, 3]. Surfaces showing, micro-, and nanoirregularities are useful in biocompatibility improvements .
Among many surface treatment technologies applied for Ti, the electrochemical one is very useful, giving surface roughening and new chemical and phase composition, which improve surface biocompatibility [5, 6, 7]. Anodic oxidation results in the formation of rough titanium oxide, which improves osseointegration. The oxidation process can be done in standard conditions as well as can be supported by spark-discharge process in the plasma electrolytic oxidation (PEO), done at high voltages [8, 9, 10]. As the results, formation of different size pores or cavities as well as nanotubes are possible [11, 12, 13]. The anodically oxidized titanium-based dental implants are commonly available . By carefully choosing the oxidation conditions it is possible to control the oxide thickness, which is correlated with its color .
During and after implantation, there is a risk of bacterial attack in the wound tissue. To avoid this inconvenient postoperative effect, an introduction of antibacterial agent into implant surface layer is highly recommended and possible. The commonly known antibacterial agents are silver ions, however it should be noticed, that high silver ion concentration could prevent osseointegration. Cytotoxicity of silver (Ag) nanoparticles at a concentration of 8 μg/cm2 to E. coli was reported previously . The large surface area to volume ratio of Ag nanoparticles provides good antibacterial behavior .
The surface, which is oxidized and has porous topography, is an excellent template for controlled introduction of silver ions. Previously  we show preferential deposition mechanism of Ag nanodendrites, which are deposited inside the pits in the oxide layer. Thus we suggest that the surface pits positively effect on both, osteoblast cells and antibacterial nanoparticle attachment.
The aim of this study was to determine of biocompatibility in vitro of the Ti, which was anodically oxidized at high voltages with additionally deposited Ag in the form of nanodendrites. Biocompatibility of the tested material was referred in relation to human osteoblasts and gingival fibroblasts. They are the major cellular elements that determine the osseointegration and the acceptance of implant in the oral cavity.
Materials and methods
The commercially pure titanium (CP-Ti) with purity >99.6 % (Goodfellow) was used for electrochemical treatment and biocompatibility test. The original ϕ10 mm Ti rod was cut into a form of small tablets (10 mm diameter and 5 mm height), grinded up to a 1000 sand paper and then polished in Al2O3 suspension to a mirror-like surface. The samples were anodically oxidized in home-made Teflon electrochemical cell. The platinum (Pt) electrode was applied as reference electrode. The oxidation process proceeded under Atlas Sollich high voltage potentiostat (300 V/3 A) control. The structural and morphological changes with using broad anodic oxidation voltages were studied in our previous works [19, 20]. In this work for biocompatibility test, samples optimally oxidized at 210 V versus OCP (open circuit potential), at constant time of 30 min were chosen. As the electrolyte, solution of 2 M H3PO4 with addition of 1 wt% HF was used. After the oxidation process, the surfaces were rinsed in water and dried under a stream of nitrogen. For obtaining antibacterial characteristics, the surface was electrochemically modified by deposition of Ag nanocrystals, in the form of nanodendrites (nano-trees) using the electrolytic deposition process . The aqueous solution of 0.01 M AgNO3 + 0.01 M HNO3 composition was served as a substrate for the Ag ions. The Ti samples were immersed in the electrolyte and additionally an Ag plate (8 cm2) was used to support the transport of the Ag ions and Ag refilling into the electrolyte. An SCE electrode was used as the reference electrode. EG&G electrochemical cell was applied and the electrolyte inside was mixed using magnetic stirring (250 rpm). The process was controlled by a Solartron 1285 potentiostat. The Ag nanodendrites depositing parameters were as follows: potential −1 V versus OCP, time 60 s, temperature 20 °C. After deposition, the samples were rinsed in distilled water and dried in a stream of nitrogen. For the purpose of this study, materials CP-Ti, anodically oxidized Ti, anodically oxidized with deposited nano-Ag are named as A0, C1, and C2, respectively.
Structure was determined using Panalytical Empyrean XRD with CuKα1 radiation, equipped with crystallographic database. Surface topography was determined using Tescan SEM model Vega 5135 equipped with PGT model Prism 200 Avalon EDS, Leica DCM 3D confocal microscope with EPI 20X-L objective, Quesant Q-scope 250 AFM. The AFM tapping mode was applied during surface scanning. The Nanoandmore probes (with pre-mounted Nanosensors SupersharpSiliconTM) were used in the surface scanning.
In vitro biocompatibility studies
Established line of human gingival fibroblasts HGF-1 (ATCC® CRL-2014™) and human osteoblast U-2 OS (ATCC®HTB-96™) were used for the study. Cell lines were derived from ATCC collection. HGF-1 cells were cultured in DMEM/Ham’s F12 media (mixed in 1:1) with l-glutamine and 15 mM HEPES, supplemented with 10 % fetal bovine serum (FBS) and 1 % antibiotic solution (10,000 U penicillin, 10 mg/ml streptomycin, 25 mg/ml amphotericin B). U-2 OS cells were cultured in DMEM medium only supplemented as described above. Cells were cultured under standard conditions, in plastic plates in an incubator at 37 °C temperature, in atmosphere of 5 % CO2 and increased humidity level of 95 %. When the cells reached confluent monolayers (~90 % of cells), the culture media were removed and cells were washed with phosphate-buffered saline (PBS). After removing PBS, the cells were harvested from the surface of plates using a 0.25 % Trypsin–EDTA. The cells were counted using a Fuchs-Rosenthal’s hematologic camera. Thus prepared cells were placed on the surface of the tested samples.
In vitro evaluation
Before testing, the samples of the material were sterilized using an autoclave at 120 °C for 15 min. Sterile samples were placed in 24-well culture plates, pre-filled with 1 ml of culture medium. Approximately 3 × 104 of cells were placed directly on the surface of studied material samples. Cultures were grown for 72 and 96 h. To ensure sterile conditions during the analyses, a chamber with laminar air flow and disposable sterile equipment were used.
Imaging of samples using fluorescence microscopy
HGF-1 fibroblasts and U-2 OS osteoblasts growing on the tested materials were imaged with a fluorescent microscope after 96 h. Cells were stained with a thiazole orange (TO). This fluorescent dye penetrates the cell membrane of living cells and bind RNA. Microscopic images of cells growing on the tested samples were archived within 10 min with a fluorescence microscope using appropriate color filters, at a magnification of 40, 100, 400, and 1000 fold.
Cell viability assay (MTT cytotoxicity test)
Evaluation of a cell cycle
After 72 and 96 h of culture, cells covering the material samples were transferred to new plastic tubes. Next, the cells were detached from the surface of the samples with trypsin. Harvested cells were resuspended in a fresh DMEM medium and centrifuged at 1000 rpm for 5 min at room temperature. After discarding, the supernatant cells were washed once with PBS. Finally, to the cell pellets a solution of propidium iodide (PI) and RNase was added, and then incubated for 1 h at 37 °C in the dark. Cells stained with PI were evaluated using the flow cytometer FACSCanto (BD Biosciences). Histograms obtained during the evaluation were analyzed by FACS Diva software. During the analysis, the mean fluorescent intensity (MFI) of stained cells was measured. The percentage of cells in S-phase of the cell cycle corresponds to proliferative activity of the cells grown on the studied material. In addition, on the basis of the cytometric histogram results, the estimated percentage of dead cells as well as cells in the G2/M phase, preceding the process of mitosis, were determined.
3D roughness parameters measured on anodically oxidized surface
3D roughness parameter
Method of measurements
In vitro evaluation results
Fluorescent imaging of samples
Cell viability assay (MTT cytotoxicity test)
Evaluation of the cell cycle
The main task is to adjust the structure of the material to achieve increased migration and proliferation of cells involved in the osseointegration of the implant. It is important to create material with high durability and resistance to the environment surrounding the tissue and a low susceptibility to colonization by pathogenic microorganisms. However, till now materials having characteristics identical to natural has not been created. It has been proven that the porous materials in the most accurate way fill these assumptions [5, 9].
The anodic oxidation process done at high voltages is very useful in surface biofunctionalization, i.e., formation of porous, rough, and biocompatible oxide [8, 9, 10, 19, 20]. As we have shown in this work, the PEO followed by spark discharge, which proceeds on the Ti surface, results in the formation of highly connected channels in the surface of the oxide layer. The channels support not only bone cell nucleation and growth, but vascularization process too. In this way formation of strong bond between implant material and bone is provided. Thus the process done at high voltages significantly overcomes the conventional low voltage oxidation process . Additionally inside the channel structure, the oxide thickness at pore bottom is lowest, which positively acts on silver ions flow from electrolyte into the surface of highest electrical conductivity (lowest oxide thickness) . The Ag nanodendrites grow inside the pores and forms biocompatible layer with enhanced bacterial killing properties. Moreover the Ag nanoparticles fixed inside the pores are protected against their removing during implant handling.
The main reason for implantation failure is local inflammation caused by reaction of tissue on the introduction of a foreign body. Cascade of negative events in the organism may be accompanied by the activity of pathogenic microorganisms. Disrupted relationship between host and microflora resulting in disease of the oral structures, mainly include dental caries, gingivitis or periodontitis or peri-implantitis . Peri-implantitis around osseointegrated implants initiated by bacterial infection affect the soft and hard tissues and can lead to bone damage . Some bacteria attached to implanted restorative materials form the biofilm that protects them against antibiotic treatment, leading to antibiotic-resistant periprosthetic infection .
Nanostructured biomaterials exhibit exceptional mechanical and surface features . They can mimic the bones, by what are considered the next-generation materials. . The shape, spatial and chemical structure, roughness, and surface energy have impact on the adhesion and proliferation of cells. However, the rough surface of the dental implants can promote the colonization by pathogens . Therefore, the next challenge for the dental implantology is an indication of materials, which will limit the risk of colonization with pathogenic microorganisms. Due to proven antibacterial properties Ag nanoparticles are intensively investigated as a material used in dental applications, mainly as dental fillings or implants [28, 29]. Strong antibacterial features of Ag are associated with the ability of this metal to interact with sulfhydryl groups of proteins as well as with DNA . Ag nanoparticles have activity against Gram-positive and Gram-negative bacteria, fungi, and viruses [31, 32].
However, the use of Ag nanoparticles carries the risk of cytotoxicity causing tissue destruction and local inflammation. Certain Ag compounds exhibited nanotoxicity for HGF . Ag nanoparticles by induction of oxidative stress can be also mutagenic for cells . However, the use of Ag nanoparticles coated on a titanium core seems to be very useful in dental technology. Mei et al.  used processes of anodization and Ag plasma immersion ion implantation for production of Ti nanotubes containing Ag nanoparticles. The technology used has confirmed the high biocompatibility of Ti and the antimicrobial effect of the Ag particles, enhanced by the large depth of the deposited Ag. Other group indicated an anti-biofilm effect of Ag nanoparticles immobilized on Ti against Staphylococcus epidermidis. This activity was related with inhibition of bacteria adhesion and down regulation of transcription of the intercellular adhesion operon (icaAD) . Liu et al. proved that Ag nanoparticles promoted proliferation and maturation of preosteoblasts and induced osteogenesis of rat bones, accompanied with suppression of bacterial survival .
In our study we examined the biocompatibility of the samples of anodized Ti modified with deposited Ag nanodendrites. Material samples were evaluated based on contact with the HGF and osteoblasts (U-2 OS) cell lines in vitro. Evaluation of fibroblasts and osteoblasts growth served as a model showing the behavior of cells of the soft and hard tissue in contact with the tested material. Cytocompatibility of tested nanosilver–titanium composites was compared to conventional microcrystalline Ti, which served as a reference material. Studies on dental implants are mainly focused on assessing the interaction between materials and bone cells as well as soft tissue, because the process of osseointegration may be disturbed by anomalies and infections of soft tissue . To evaluate the cytocompatibility of the tested samples, in vitro characterization tests in static condition were performed. Samples were tested for cytotoxicity with MTT assay. Proliferative activity of cells directly growing on samples was determined with cytofluorimeter (determination of the percentage of cells in S-phase of the cell cycle stained with PI). Furthermore, cells growing on the tested samples were stained with thiazole orange and visualized in a fluorescence microscope. Our analyses indicated that the nanocrystalline Ti modified with Ag has a higher degree of biocompatibility in comparison with the unmodified reference material, which was microcrystalline Ti. Cells used in the tests showed a higher relative viability potential in contact with tested materials, than cells in contact with the surface of control samples. Both, fibroblasts and osteoblasts showed numerous NOR within the cell nuclei what may serve as an evidence of increased proliferative activity of tested cells. This observation was supported by numerous cells in the process of mitosis. Moreover, cytometric analysis confirmed these results, particularly when tested cells showed the stable level of proliferation activity and lower rate of mortality.
Modification of the titanium through the anodic oxidation process at high voltages with additional deposition of nanosilver can significantly change the properties of the reference material, which can improve its biocompatibility, but also can introduce new quality. The development of such material is a major goal of modern materials science. Anodized titanium modified with deposited silver nanodendrites, the material presented in this report significantly brings us closer to this target.
The work has been financed by Polish Ministry of Science and Higher Education within statutory activity.
Compliance with ethical standards
Conflict of interest
Authors declare no conflict of interest.
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