Biocompatibility of Titanium Oxide Nanotubes Layer Formed on a Ti-6Al-4V Dental Implant Screw in hFOB Cells In Vitro

Abstract

The surface modification of dental implants with nanostructured films enables the development of the next generation of biomaterials that promote osseointegration. In this study, a uniform layer of titanium oxide nanotubes (TNTs) was successfully formed on a Ti-6Al-4V dental implant screw through anodic oxidation. TNTs were morphologically characterized by Scanning Electron Microscopy (SEM), obtaining dimensions of 64.88 ± 10 nm in diameter and 5.34 ± 5 µm in length. Additionally, a crystal size of 23.45 nm was determined by X-ray diffraction (XRD) analysis. The TNT layer on the dental implant screw was evaluated in an in vitro system in direct contact with human osteoblast cells (hFOB) for 24 h and 48 h, finding cell growth near to the screw threads. Further, the biocompatibility of the dental screw coated with TNTs was evaluated using a flow cytometric assay with 7-AAD, demonstrating that cell viability was not affected at 24 h and 48 h. This study opens the perspective of the study of inflammation and osseointegration induced by implants coated with TNTs.

1. Introduction

Titanium (Ti) and its alloys are still the reference for biomaterials due to their biocompatibility, corrosion resistance, and mechanical properties. The most important titanium alloy is Ti-6Al-4V, which has been used for biomedical applications, including orthopedic implants such as knee and hip replacements, spinal fusion devices and dental implants [1]. Another important property is the elastic modulus of Ti-6Al-4V and bone are 116 GPa and 20 Gpa, respectively. Additionally, Ti-6Al-4V exhibits a better elastic compatibility with bone to other orthopedic and dental alloys such as AISI316L, with an elastic modulus of 190 Gpa [2,3]. Thus, this elastic compatibility promotes better load distribution between the implant and the bone, avoiding a possible mechanical failure caused by the reduction in bone density [4].

Moreover, a thin titanium oxide layer is formed when Ti is exposed to air, which makes it biocompatible [5]. However, due to its smooth nature, this layer does not allow proper osseointegration or osteoconductive and osteoinductive properties when it is used as an implant [6]. Therefore, functional interaction between the bone and the implant surface is important, which is a complex phenomenon that depends on stable osseointegration. Efficient osseointegration is a dynamic process, wherein cells secrete various cellular factors to promote osteogenic cells and induce differentiation [7,8]. For example, titanium screws implanted after fracture easily fall off in osteoporosis patients. For this reason, it is of great clinical importance to find an effective method to modify the implants’ surfaces in addressing bone screws loosening [9].

A variety of coating techniques such as physical vapor deposition (PVD), chemical vapor deposition (CVD), electrochemical modification, or plasma electrolytic oxidation exist, allowing for the surface modification of biomaterials [10]. Currently, the modification of titanium as a biomaterial is being investigated worldwide to obtain nanostructured surfaces. The nanostructure of titanium oxide coatings has been proved to play a crucial role in enhancing surface roughness and hydrophilicity, both of which are key factors in regulating biological events, including cell attachment and protein adsorption. Additionally, the porosity and large surface area provide a barrier to protect the implants from the external environment [11]. TNTs can also be used for local drug delivery, such as anti-inflammatory or antibiotic compounds, to enhance the clinical function of the implants [12]. Hence, anodic oxidation is a process that has been used due to its ease of handling and the formation of a highly ordered TNT layer, which promotes the adhesion and proliferation of osteoblasts [13,14,15,16].

The morphology of the anodized layer can be affected by voltage, temperature, time, anodizing distance, and electrolyte composition. There are related studies on the electrolyte composition, such as the concentration of fluoride ions, the type of solvent, and the variation of water content, which affect the growth rate of the nanotubes by changing the current of the anodizing process [17]. Consequently, TNTs serve as a precedent for the design of the next generation of orthopedic and dental implants, ensuring that they maintain biocompatibility and promote osseointegration. However, a less-studied condition is the shape of the surface where the nanotubes will be formed, as the orthopedic and dental devices are designed with sharp curved surfaces, such as screw threads [18]. Luo et.al. mention that the formation of TNTs on the surfaces of veterinary fixation screws and their evaluation through a fretting test ex vivo on a sheep femoral bone presents a challenge because the areas of greatest wear are the threads, which finally induce mechanical failure [19]. Therefore, this study aimed to form TNTs on human dental screws and evaluate them in vitro in direct contact with human osteoblasts to confirm the preservation of the biocompatibility of the modified surface.

2. Materials and Methods

2.1. Anodic Oxidation

TNTs were formed by anodic oxidation on dental implant screws of Ti-4Al-6V (ASTMB348 Gr5, Quimica Islas, Mexico). The dental screws’ dimensions were 1.93 mm in diameter and 15.20 mm in length. First, the screws were pre-treated through a metallographic and polishing process. Subsequently, they were cleaned ultrasonically for 5 min. The anodizing process was performed in an electrolytic cell in which the dental implant screws were used as the anode and a graphite electrode was used as the cathode, with a separation of 1 cm between them (Figure 1).

The electrolyte was based on ethylene glycol (EG) with 99.8% purity, 1 wt.% of distilled water, and 0.5 wt.% of ammonium fluoride (NH4F) with 99.98% purity (conductivity and pH: 1.23 mS cm⁻¹ and 7.4, respectively) (Sigma-Aldrich, St. Louis, MO, USA). The anodizing time was set to 12 min per side and a constant working voltage of 60 V was applied in order to achieve a uniform titanium oxide nanotube layer along the screws. These experimental conditions were chosen on the basis of the favorable results obtained in a previous published study [20].

2.2. Characterization

The structure of the TNT layer was obtained by an XRD-D8 Focus diffractometer (Bruker, Billerica, MA, USA) with Cu-Kα radiation (=1.5418 Å). The results were analyzed using Hull and Davey’s graphical method and Scherrer’s equation. Moreover, the presence and morphology of the nanotubes were characterized by 250 FEG SEM (FEI Quanta, Hillsboro, OR, USA). SEM micrographs enabled image processing to obtain the diameters and lengths of the nanotubes with the assistance of Image-Pro software 11.

2.3. In Vitro Assay Conditions

2.3.1. Cell Cultures and Maintenance

A human fetal osteoblast cell line (hFOB 1.19) was cultured in accordance with ATCC recommendations in a 1:1 mixture of Ham’s F12 Medium and Dulbecco’s Modified Eagle Medium (DMEM-F12) (Corning), supplemented with 2.5 mM L-glutamine, 10% fetal bovine serum (Corning), and 1% penicillin–streptomycin (Corning) to form a complete medium. These cells were cultured at 37 °C in an atmosphere of 90% humidity with a supplement of 5% CO₂. At the 4th passage of the cell culture, the cells detached from their culture plate, and a cell count was performed to achieve a cell density of 20,000 cells per mL of culture medium for all experiments. Additionally, micrographs of the osteoblast cells in direct contact with the anodized dental implant screw were obtained using an optical microscope (MOTIC AE-30, Xiamen, China).

2.3.2. Cell Proliferation and Viability Test by Flow Cytometry

Dental implant screws underwent anodization, followed by three cycles of ultrasonic washing, each lasting 5 min. Subsequently, two cycles of washing with 70% ethanol and one cycle with sterile double-distilled water were performed. The screws were then sterilized using UV-C light (254 nm) for 90 min before being placed in 12-well culture plates. Furthermore, UV-C radiation sterilization does not affect the structure of the nanotubes formed on the surface of the screws [21]. Cell counting was conducted by processing images of the cell–screw interaction using a self-written Matlab code (The MathWorks Inc., Natick, MA, USA). The script identifies regions of interest by mapping centroids and defining their boundaries through contour detection.

The viability of the screws was evaluated using the 7-aminoactinomycin D (7-AAD, BioLegend) reagent , comparing four experimental conditions: Negative control: cells without stimulation (NS), direct contact of the cells with anodized screws (nanotubes), direct contact of the cells with screws without anodization (no nanotubes), and the positive control of cells treated with 15% dimethylsulfoxide (DMSO, Sigma-Aldrich). In each well, 800 µL of the culture medium containing a homogeneous cell suspension was added. The suspension was pipetted directly and uniformly over the implant’s surface, allowing cells to settle and adhere, and the implant was placed horizontally, covering >10% of the well’s surface area as recommended by ISO 10993-5 to maximize cell–material interaction. After the culture period, cells were collected in 5 mL round-bottom test tubes (Falcon), washed with 3 mL of 1X PBS, and centrifuged at 1500 rpm for 5 min. Subsequently, in a staining volume of 500 μL of 1X PBS, 0.5 μL of 7-AAD reagent (BioLegend) was added, and the samples were incubated at room temperature for 10 min, protected from light. Immediately following the incubation period, 10,000 single events were acquired using an Attune flow cytometer (ThermoFisher). Data analysis was performed using FlowJo™ V10 software.

3. Results

3.1. TNT Characterization

3.1.1. XRD Pattern

The presence of Ti, Ti₂O, and TiO are observed in the XRD pattern of TNTs formed by anodic oxidation (Figure 2). The main diffraction peak corresponds to the crystalline hexagonal structure of Ti₂O at 2θ = 40.45, which is slightly shifted to the right compared with the peak representing Ti at 2θ = 40.182. Although titanium also has a hexagonal close-packed structure like Ti₂O, the presence of oxygen affects its atomic arrangement.

The calculated lattice parameters of the Ti₂O are a = b = 2.934 Å and c = 4.850 Å, which produces a variation in the c/b ratio = 1.653. The average crystallite size is 23.45 nm, which was determined by Scherrer’s equation (1), where k is a constant (0.9) and β is the full width at half-maximum height of the diffraction peak:

$$D_{hkl}=\frac{k\lambda }{\beta \cos \left(\theta \right)}$$

(1)

3.1.2. Morphology

In Figure 3a, an intact TNT layer can be observed on the dental screw, which was positioned perpendicularly to the electrode functioning as the cathode, with an anodizing time of 12 min per side. In contrast, the partially detached TNT layer shown in Figure 3b was obtained by anodizing for 12 min on only one side. Therefore, the SEM images obtained in Figure 4 represent a close-up of the modified screw in Figure 3a, which has a uniform nanotube layer on the titanium alloy surface.

The SEM image (Figure 4a) exhibited some detached patches in the TNT layer, which is related to the β-phase of the Ti-6Al-4V alloy, indicated by yellow arrows. This is consistent with the EDS analysis (Figure 4c) conducted in the area within the blue rectangle, where the peak representing vanadium is larger than the aluminum peak, with relative concentrations of 5.38% and 2.88%, respectively. The β-phase is more ductile than the α-phase, allowing for greater deformation capacity before fracturing, but it is less resistant to fatigue. Therefore, it was not possible to obtain a completely uniform layer due to the high solubility of vanadium oxides [22].

The anodized dental screw exhibited a well-defined array of TNTs (Figure 4b) with an average length of 5.34 ± 5 µm. In Figure 4d, the top part of the nanotubes with well-defined walls can be observed, along with the measurement of a nanotube with an inner diameter of 69.2 nm. Meanwhile, Figure 4e presents a graph of the diameter range from 50 to 90 nm obtained through image processing. However, the most frequent diameters observed are 50 and 60 nm, with an average diameter of 64.88 ± 10 nm. This diameter is similar to that reported in a previous study [12], as maintaining a F-concentration between 0.3 wt.% and 0.7 wt.% induces intense chemical dissolution of TiF6, resulting in diameters ranging from 30 to 70 nm [23].

3.2. In Vitro Assay

Proliferation and Viability of hFOB Cells with TNTs on a Dental Screw

The number of cells interacting with the anodized screw shows a marked rise (Figure 5c), with cell counts doubling over this period. Specifically, at Time 0, the cell count was 17,192 ± 693; at 24 h, it increased to 34,313 ± 2561; and by 48 h, the count reached 56,152 ± 5519. Throughout the duration of direct interaction with the anodized dental screw, the hFOB cell line exhibited consistent growth and maintained a stable morphology. These results indicate that the anodized material does not release residues that adversely affect cellular morphology or impede cellular proliferation.

To assess whether the modified material affected the hFOB cell line, a viability test evaluating membrane integrity was conducted using 7-AAD, a DNA intercalating dye, analyzed by flow cytometry. Cells were incubated with the device for 24 and 48 h, during which, membrane integrity was preserved, indicating viable cells, as determined by the size (FSC-A) and granularity (SSC-A) of the population (Figure 6a).

In contrast, cells treated with DMSO exhibited changes in morphology and population density (Figure 6a, right bottom dot plot image). In viable cells, 7-AAD does not penetrate the membrane, preventing non-covalent binding to DNA and thus resulting in no fluorescent intensity, as shown by the histogram’s shift to the left. In contrast, in DMSO-treated cells, the dye binds to DNA due to membrane damage, producing a fluorescent peak shifted to the right (Figure 6b). Dot plot analysis was used to determine the percentage of viable cells at different time points (Figure 6c,d), showing no change in cell viability in the presence of the dental screw.

4. Discussion

The novelty of this work lies in the formation of TNTs on the surface of dental implant screw based on the experimental conditions of our research group’s results [12], using an accessible and easy-to-implement technique. This contrasts with other techniques that rely on titanium sheets coated with a photoresist using a spin coater and laser lithography, which increases production costs [24]. The experimental conditions of this study enable reproducible responses consistent with previous works, such as the Ti₂O obtained during the formation of TNTs. This type of oxide has only been reported and published in in vitro evaluation systems by our research group, with the important fact that it maintains crystallinity despite having a low oxygen content. The lattice parameters are similar to those reported in the references, which are a = b = 2.960 Å and c = 4.830 Å, with a larger variation of the c/b ratio = 1.631, resulting in an error percentage of 0.8% for a and 0.4% for the c lattice parameters. Additionally, the variation of the c/b ratio indicates the presence of interstitial oxygen [25].

On the other hand, it was observed that the optimization conditions for the TNT layer formed by anodic oxidation depend on the constant voltage, oxidation time, current field, and the ion flow. Consequently, in the screw anodized for 12 min on one side only, partial detached of the layer occurred because a proper ion flow was not established on the side farthest from the graphite electrode, unlike when anodizing both sides. Furthermore, the research by Tomastik et al. demonstrates that thin films, such as TNT coatings, gradually thin until the substrate is exposed, similar to the behavior of soft coatings on hard substrates [26]. Pereira et al. exhibited that the nanotube layer formed by anodic oxidation frequently presents adhesion failures, which is a separation between the coating and the substrate [27]. However, the TNT layer’s obtained length falls within the reported range of 200 nm to 1000 µm in studies of the anodic oxidation process [28]. This is consistent with other works that mention achieving lengths close to 500 nm in strong HF acids [23] and extends to 6.4 µm in fluoride solutions [29].

The presence of a nanostructured surface has been shown to have a positive impact on cell adhesion and proliferation, as it increases the contact area, enhances cell–material interaction, promotes the adsorption of key proteins for cell adhesion, and modulates the biological response, creating a more favorable environment for cell growth [30,31]. Currently, various studies have not guaranteed cell growth and adhesion on nanostructured biomaterials. This is due to the shape of dental or orthopedic implants, as well as the nature of the modified surface. Our group previously conducted research using a device designed to allow direct contact, under patent registration number MX/a/2019/006316 [12]. However, to assess cell adhesion in the configuration of a dental screw, a series of modifications to the patent are required to achieve complete cell growth on the entire surface.

Because the cells adhered to the well base and the vicinity of the dental screw, we decided to evaluate the cell viability. This study demonstrates the biocompatibility of TNTs in a human cell line using flow cytometry, which has become a frequent tool to assess the compatibility of nanomaterials [32]. 7-AAD is a DNA intercalant that allows discrimination between live and dead cells by emitting stable fluorescence, an advantage over propidium iodide (PI), whose emitted fluorescence is less stable. With this assay, we demonstrated that the presence of the dental screw is not toxic to the hFOB cell culture, which is consistent with optical microscopy, where even more cell growth is observed near the dental screw coated with TNTs. However, since both 7-AAD and PI are DNA intercalants, they detect cells whose membrane has been compromised, which is typical in necrotic cells [32,33]. To evaluate apoptotic cells, markers such as Annexin V are used, which binds to the extracellularly exposed phosphatidylserine during apoptosis. However, the use of trypsin during cell processing can temporarily damage the cell membrane and overestimate the number of cells in apoptosis that are not truly apoptotic [33]. Therefore, perspectives for evaluating the compatibility of the TNT-coated dental screws include measuring cellular apoptosis using caspase expression assays, as well as markers for stress, inflammatory mediators, and oxidative stress such as reactive oxygen species (ROS), and confirming that hFOB cells grow closer to the dental screw coated with TNTs by measuring their cell cycle.

5. Conclusions

To sum up, morphological characterization demonstrated that a uniform TNT layer was obtained when anodizing both sides of the screw. Moreover, the structural analysis revealed the presence of Ti₂O, which is a type of oxide that has only been reported by our research group as a modified biomaterial alongside its in vitro evaluation.

With the viability assay, we demonstrated that the presence of the TNTs on the dental screw is not toxic to the hFOB cell culture, which is consistent with optical microscopy, where even more cell growth was observed near the dental screw coated with TNTs. This study opens the perspective of the study of new cellular mechanisms induced by implants coated with TNTs such as inflammation and osseointegration.