HOS cell adhesion on TiO2 nanotubes texturized by laser engraving

Due to its outstanding properties, the titanium and its alloys have been widely used in the dental and orthopaedic fields as biomaterials. The TiO2 nanotubes surface and the texturized process by laser engraving enables significantly accelerated osteoblast adhesion on the biomaterial. For this reason in this paper, the HOS cell responses on TiO2 nanotubes fabricated on Ti6Al4V alloy and texturized by laser engraving were evaluated. The test surfaces were carried out on smooth Ti6Al4V as control, TiO2 nanotubes (NT) and surfaces with micropoints obtained by laser engraving, with 1mm spacing (NTP1) and 0.5mm (NTP2). The results show that the texturized process enables decreases the contact angle thus improving wettability of the TiO2 nanotubes surface. The NTP1 and NTP2 surfaces show excellent cell adhesion and spreading on the surface, which is evident in epifluorescence microscopy images. Furthermore, the NTP1 and NTP2 surfaces improved the cell proliferation at 18% and 16% respectively in relation with NT surface, showing that the laser texturing improves cell response of TiO2 nanotubes.


Introduction
The biomaterials were developed in order to fulfill needs that could not be met naturally [1], for this reason this materials are used in a large number of applications, bone implants, joints, vascular stents, skin substitutes, plastic surgery, cranial and dental implants among many others [2][3][4]. Bioactive materials used in orthopaedic implants to improve the integration of the implant to the bone and enhanced osseointegration process [5,6]. One of the most widely used materials for this purpose is titanium and its alloys, Ti6Al4V alloy mainly because of their excellent properties.
Currently different methods are being studied to improve the surface properties of titanium and its alloys in order to achieve and accelerate the osseointegration process in the surface of these materials. One of the most widely used methods has been the electrochemical anodization process [7][8][9]. This method allows grow controlled the titanium oxide layer, which is formed naturally in the surface of this material. Modifying anodizing parameters, it can be generated different morphologic surfaces and properties that can improve the material behaviour to be implanted in the human body. Another method for modifying the surfaces of orthopaedic implant materials is the textured by laser engraving [10][11][12][13][14]. This method has improved the cell adhesion of Ti and Ti6Al4V surfaces by creating patterns on surfaces. These patterns become places where a preferential cell adhesion occurs, which increase the adhesion of osteoblast cells.
Therefore, in this work we study the effect of laser-textured surface in the cellular response of TiO2 nanotubes grown on Ti6Al4V.

Sample preparation
Ti6Al4V cylindrical disc with 14mm diameter and 3mm thick was grinding with silicon carbide paper in successive grades from 220 to 1200 grit and subsequently the disc were cleaned by sonication in ethanol and deionized water for 15 minutes. Some these substrates were anodized to get TiO 2 nanotubes, theses substrates were immersed in a dilute acid mixture of nitric acid (HNO3) and hydrofluoric acid (HF) for 1 minute in order to remove the thin oxide layer that spontaneously forms on the Ti6Al4V surface in presence of the air. The anodization process was carried out in a conventional two electrodes cell, using a solution of 0.5wt% HF, applying 10V during 1 hour at room temperature. Fourth different surfaces were used in this study. They include untreated surface (Ti6Al4V), TiO 2 nanotubes (NT TiO 2 ), and laser engraving with two different circular patterns (NT TiO2 P1 and NT TiO 2 P2). The patterning surfaces were obtained by laser engraving using a methodology previously described in [15] using a sealed CO 2 laser with maximum power of 40W. The circular patterns generated on the surface can be seen in the Figure 1. All samples were sterilized in autoclaved at 120°C for 20 minutes and then by UV radiation for 30 minutes on each side.

Biological test
These test were performed using a methodology previously described in [15]. For this, HOS cells was cultured in 25cm 2 Falcon culture plates and maintained in an incubator at a temperature of 37°C regulated with 5% CO 2 , 95% air, and saturated humidity. RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin was used as the cell-cultured medium. For adhesion assay, 2.5×10 4 cells/ml in cultured medium were placed on each material and incubated at 37°C. After 1, 3 and 5 days the cells were detached using 0.5ml Trypsin-EDTA for 5 minutes and counted microscopically in a Neubauer chamber. In addition a fluorescence staining were made directly in each sample using Hoechst 33342 fluorescent stain and epifluorescence microscopy (Nikon Eclipse E4000), B-2A filter (Ex=450-490, DM=500, Ba=515). Images were obtained using a camera Nikon Coolpix 5000. UV2A. Each time experiments were performed in duplicate and the results were calculated as number of cells per surface area.

Results and discussion
The TiO 2 nanotubes morphology grown on Ti6Al4V alloy by applying 10V in bath containing 0,5% HF concentration is shown in Figure 2. The nanotube inner diameter in the mouth is approximately 30nm. The formation of nanotubes preferentially grown in the alfa phase along with beta phase, besides dissolution of the oxides was observed. Such morphology has been widely described for TiO 2 nanotubes grown in acid electrolytes with F containing [16,17].
As shown in the Figure 3, the patterns has a semicircular shape with diameter of 150µm, the separation spots on the vertical axes is about 1250µm and the horizontal axes is 1295µm for NT TiO2 P1 and 630um for NT TiO 2 P2 respectively. This variation in the dimensions of the patterns was previously described in [15] and the movement performed by the laser engraving equipment to achieve surface texturing the surface attribute. The hydrophilic/hydrophobic behaviours of the surfaces are shown in the Figure 4. Ti6Al4V smooth surface have a hydrophilic behaviour while the anodized surface had a hydrophobic behaviour. This hydrophobic behaviour has been widely reported [18,19]. The TiO 2 nanotube surfaces texturized by laser engraving (NT TiO 2 P1 and NT TiO 2 P2) show a reduction in a contact angle, making these anodized surfaces less hydrophobic. Previously it reported that the laser texturing process on smooth Ti6Al4V, TiO2 nanotubes and TiO 2 nanotubes texturized by laser engraving result non-toxic to HOS cells, this results consistent with previously reported in the literature [20][21][22]. Moreover, as shown in Figure 5, the HOS cell adhesion show a high affinity to surfaces with TiO 2 nanotubes. The cell adhesion process is favoured on these surfaces due to the roughness and increased surface area generated by these nanometrics structures [20,22]. The Figure 6 shows a representative images of the HOS cells during three incubation times, as can be seen the HOS cells were randomly oriented and well dispersed. On the TiO 2 nanotube surface at 5 culture days the HOS cells days is possible appreciate.

Conclusions
This study produced nanotube arrays with a uniform tube diameter through the anodization of Ti6Al4V. These surfaces were texturized by laser engraving. In cell culture experiments, the nanotube and nanotube texturized surface exhibited adhesion and spreading that was more rapid than that of untreated surface, despite a higher contact angle. These results provide evidence that nanotube texturized can significantly enhance cell activity. It was shown that the texturized by laser engraving is an effective means of promoting cell functions on nanostructured surface implants, thereby improving the bone-implant interface in vivo.