Comparison of Fibroblast and Osteoblast Response to Cultivation on Titanium Implants with Different Grain Sizes

1Department of Medical Chemistry and Biochemistry, Faculty of Medicine in Pilsen, Charles University in Prague, Karlovarska 48, 301 66 Pilsen, Czech Republic 2Department of Biology, Faculty of Medicine in Pilsen, Charles University in Prague, Alej Svobody 76, 323 00 Plzen, Czech Republic 3Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Alej Svobody 76, 323 00 Pilsen, Czech Republic 4Department of Applied Electronics and Telecommunications, Faculty of Electrical Engineering, University of West Bohemia, Univerzitni 26, 306 14 Pilsen, Czech Republic 5Department of Stomatology, Faculty of Medicine in Pilsen, University Hospital and Charles University, Alej Svobody 80, 301 00 Pilsen, Czech Republic


Introduction
Titanium is commonly used for dental as well as orthopaedic implants due to its properties such as biocompatibility, nontoxicity, and corrosion resistance.[1][2][3][4][5][6][7][8][9][10].However, commercially pure titanium (cpTi) has excellent biocompatibility but relatively poor strength, and titanium alloys have superior strength but they contain potentially toxic or allergic ingredients [11,12].Long-term stability of the titanium implants is related to their wear resistant properties.Loosening or failure of the implant can be caused by inflammation and bone resorption induced by the wear debris in the form of titanium particles derived from the implants entering into the surrounding tissues [13].Therefore, it is essential to improve the biocompatibility and wear resistance of a titanium implant for its successful long-term survival [14].
An important direction intensively developed in recent years is the investigation of mechanical properties of nanostructured materials.The formation of nanostructures in metals leads to higher strength.There is great interest in the processing of bulk, fully dense nanostructured metals and alloys.The fabrication of such materials based on severe plastic deformation (SPD) methods seems very interesting and useful.The first developments and investigation of nanostructured materials processed using SPD methods were carried out by Valiev and colleagues more than two decades ago [15][16][17].The method for large plastic deformations and formation of nanostructures in our study was equal channel angular (ECA) pressing.The ECA pressing method was carried out by deformation of massive billets via pure shear.Its goal is to introduce an intense plastic strain into materials without changing the cross-section area of billets.Due to that, their repeat deformation is possible.The method was further developed and applied as an SPD method for processing structures with submicron and nanometric grain sizes.Nanostructured titanium produced by SPD processing binds together the advantages of aforementioned materials, that is, excellent biocompatibility and extraordinary mechanical properties [11,18].There are not many companies producing bulk nanotitanium and the cost of this material is very high, approximately 10 times higher than conventional titanium [19].
It is known that nanostructuring of material changes its biological properties compared with material of the same chemical composition, but the mechanism of this phenomenon has not yet been clarified [20].The first evidence of such an effect was provided by Webster et al. in 1999, who found that osteoblast adhesion and bone formation significantly increased on nanostructured titanium surface compared with conventional titanium [21].Since that time many in vitro as well as in vivo studies have investigated the impact of the nanostructured surface on the behaviour of cells and provide evidence that key biological processes, such as proliferation, gene expression, and initial protein adsorption that control such events, can be easily manipulated by modifying the nanotopography of implants [22][23][24].It has also been proven that cells sense and react to nanotopography, by exhibiting changes in cell morphology, orientation, and cytoskeletal organisation [25][26][27].
An important issue is to improve osseointegration of an implant to its surrounding natural bone tissue [14,28].The long-term success of a dental implant depends not only on the integrity of osseointegration but also on the contact with surrounding soft tissue [29][30][31].It is well known that cellular behaviour, such as adhesion, morphologic change, migration, functional alteration, and proliferation, is determined by surface properties such as composition, surface energy, topography, and roughness [32][33][34][35].Nanotopography of an implant improves and accelerates osseointegration [19,36].Cell lines have been widely investigated as model systems to explore the influence of nanoscale surface topography on cellular response [37].
The aim of the present study was to compare nanostructured titanium with different grain size with respect to biocompatibility using human fibroblast cell line HFL1 as well as human osteoblast cell line hFOB 1.19.

Material and Methods
2.1.Materials.All the samples were obtained from commercially pure titanium (cpTi) by the ECA pressing method from cpTi grade 2. They have a cylinder shape with a diameter of 4.98-5.05mm and height of 2.93-3.01mm (Figure 1(a)).Used samples of titanium have grain sizes of 160 nm, 280 nm, and 2400 nm.For each grain size there were two types of sample: cross-section (−) and longitudinal section (+) (Figures 1(c) and 1(d), resp.).
Each implant was cleaned and sterilised before usage.The procedure contains incubation in a trypsin solution (0.25% (w/v) Trypsin-0,53 nM EDTA solution, PAA Laboratories GmbH, Austria) (30 minutes, 37 ∘ C), followed by incubation in an ultrasonic bath (20 minutes, 25 ∘ C) incubation in acetone (20 minutes, 25 ∘ C), and at the end rinse in 70% ethanol and deionised water.Finally the implants were sterilised by autoclaving.

Characterization of Surfaces.
All the sample types with different grain sizes and sections were analysed by scanning electron microscopy (SEM; JSM 6380, JEOL, Japan).Secondary electron channel was used for the observation.
The surface roughness of each sample was measured three times using a mechanical contact profilometer Surtronic 25 (Taylor Hobson, UK).Surface roughness of samples was quantified by arithmetical mean roughness Ra (defined as arithmetic average of the absolute values of the profile height deviations from the mean line) and ten-point mean roughness Rz (defined as the sum of the average value of absolute values of the heights of five highest profile peaks and the depths of five deepest profile valleys measured in the vertical magnification direction from the mean line).The surface roughness was measured at a traverse speed of 1 mm/s with a diamond-tipped stylus with 5 m radius.The average of the three measurements was recorded as the mean surface roughness for each specimen.

Cell Cultures.
Both used cell lines were obtained from ATCC (American type culture collection, Rockville, MD, USA) and cultured in accordance with ATCC recommendations.Culture media were refreshed as needed.

Cell Viability and Proliferation.
Cell proliferation after 48 hours from plating was assessed by MTT viability and proliferation assay (ScienCellTM Research Laboratories, Carlsbad, CA, USA) according to the manufacturer's instruction.This assay is based on the conversion of pale yellow tetrazolium MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to purple formazan crystals, which can be solubilised and then spectrophotometrically quantified.
The samples of implants were placed into a 96-well plate (TPP, St. Louis, MO, USA).Cells harvested with trypsin solution from Petri dishes were resuspended in culture medium and seeded at a density of approximately 500,000 cells/mL onto the top of the discs of nanostructured titanium in 20 L volume.As positive control, cells grown directly on the 96-well tissue culture plate were used.After 48-hour incubation, cells were washed with phosphate-buffered saline (PBS) and incubated with 10 L MTT (25 mg/mL) solution at 37 ∘ C.After 4 hours, 100 L of MTT solubilisation buffer (equal to the volume of original culture medium) was added to each well and the insoluble formazan formed was dissolved by pipetting up and down.The absorbance was measured at 570 nm (spectrophotometer Nano Drop 1000, Thermo Fisher Scientific, Waltham, MA, USA), subtracting the background absorbance determined at 690 nm.

Fluorescent Microscopy 2.5.1. Cells Staining. Cultured cells were stained with
CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes, Inc., Eugene, Oregon, USA) according to the manufacturer's instruction.Briefly, cells were properly washed with PBS and incubated with 4 M CMFDA working solution for 30 min at 37 ∘ C.Then, the dye working solution was replaced with fresh, prewarmed medium and the cells were incubated for another 30 minutes at 37 ∘ C. Stained cells were analysed using an Olympus IX 70 fluorescent microscope equipped with Cell R system at 40x, 100x, and 400x magnification.
The initial cell attachment and the spreading of the cells on the substrate with different grain size were examined after 6 h and 24 h, respectively.The area occupied by the cells was assessed by analysis of gained images by the programme ImageJ (W. S. Rasband, U. S. National Institutes of Health, Bethesda, Maryland, USA).

Immunocytochemistry.
The samples of implants were placed into a 24-well plate.Five hundred cells were seeded onto the top of the discs of nanostructured titanium cells and incubated for 2 or 48 hours.Fixation was performed by 3% formaldehyde in PBS for 15 min at 37 ∘ C followed by three rinses with PBS.Permeabilisation was carried out by incubation with 0.1% triton X-100 solution in PBS for 10 min at room temperature.Blocking with 2% normal goat serum (Milipore, Billerica, MA, USA) followed for 1 h at 4 ∘ C. Each sample was double stained.Indirect immunofluorescence staining was done with a mouse monoclonal antivinculin antibody HVIN-1 diluted in PBS (1 : 100) and goat -mouse Atto488 conjugated secondary antibody.For actin staining, Phaloidin-Tetramethylrhodamine B isothiocyanate (TRITC) was added into the solution of secondary antibody in PBS (0.75 Atto488 : 1.5 TRITC : 100 PBS) (Sigma-Aldrich, St. Louis, MO, USA).Incubation with primary antibody was overnight.The second incubation was 2 hours at room temperature in the dark.Samples were analysed using an Olympus IX 70 fluorescent microscope equipped with a Cell R system at 40x, 100x, and 400x magnification.
2.6.Statistical Analysis.Microscopic analysis was carried out two times at a minimum, using two samples per group.In case of MTT assay, two independent experiments with quadruplicate measurements were performed.Cell viability was compared by analysis of variance (ANOVA).If ANOVA indicated a significant difference ( < 0.05) statistical comparisons were computed by two-tailed unpaired -test with the value of significance  < 0.05.Statistical analysis was performed using the SigmaPlot 12.5 software (Systat Software Inc., San Jose, California, USA).

Sample Characterization.
Sample characterization was performed by SEM. Figure 2 shows SEM images of the titanium sample surfaces.The surface roughness quantified by arithmetical mean roughness Ra and ten-point mean roughness Rz of each sample is shown in Figure 3. Values of Ra were between 0.3 and 0.6 m and Rz between 1.5 and 3.0 m.We did not find any significant differences in surface roughness (for both parameters Ra and Rz) among studied materials ( = 0.1097 and  = 0.0623, resp.).We also analysed differences between particular materials.
The occupied area after 6 hours was significantly higher on material 160− in contrast to 280− and both types of 2400 (− and +) ( < 0.0001;  = 0.0170;  = 0.0259; resp.).Microscopic observation revealed that after 6 h, fibroblasts presented a mainly rounded morphology (Figures 5(a) and 5(c)).After 24 h the cells elongated and presented a mainly spindle-like structure.On the tissue culture plastic, we did not see any specific orientation (Figure 5(d)).However, on material samples the cells were aligned along concentric grooves (Figures 1(b) and 5(b)).

MTT Assay.
The viability of two cell lines (hFOB 1.19, HFL1) was estimated by MTT assay (Figure 6).The viability of fibroblasts growing on materials 160−, 280+, and 2400+ was significantly lower than on control plates ( = 0.026).The osteoblast viability was lower, when growing on all types of studied titanium materials with the exception of 160+ material in comparison with the control ( < 0.0001).
The medians of viability of the cells (% of positive control) are shown in Table 1.We found higher viability of osteoblasts comparing materials 280− with 160+ and 2400+ with 160+ ( = 0.0162,  = 0.0372; resp.).The comparison of other pairs of materials did not exhibit any significant differences.On the other hand, material 160+ was a significantly better substrate for culturing osteoblasts than all other studied materials ( = 0.0072).

Immunocytochemistry.
In order to compare morphology of the cytoskeleton, fibroblastic cells grown on the six different titanium materials underwent actin labelling with TRITC conjugated phalloidin and vinculin labelling with goat -mouse Atto488 conjugated secondary antibody for mouse monoclonal anti-human vinculin antibody HVIN-1 (Figure 7).
Two hours after seeding, cytoskeleton analysis showed that cells presented a round shape and were not yet spread properly on the surfaces.On all tested materials, at this point in time, focal contacts could be seen as positive spots localised at the cellular edge.
After 48 h,the cytoskeleton analysis mainly showed cells with an elongated bipolar morphology.On all tested surfaces, vinculin-positive focal contacts were present homogenously on the whole cell surface, with a slightly higher density at the cell periphery, at the ends of F-actin filaments.These data denote that the adhesion phase occurred on all tested materials.

Discussion
In our work we examined how grain size of nanostructured titanium material influences the behaviour of fibroblastic as well as osteoblastic cells grown on its surface.
The grain size was shown to be an important factor that influenced not only the strength of material but also its interactions with cells.Kim et al. proved that the ultrafine grain titanium prepared by the ECAP method had better biocompatibility concerning wettability, cell adhesion, and proliferation of mouse fibroblasts [39].Our results did not clearly prove that grain size has a distinct impact on viability or proliferation of used fibroblast model (HFL1).The only differences we saw were related to the initial phase of attachment, but until 24 hours after seeding, differences almost disappear.We saw faster cell attachment on material with the smallest grain size in examination.
The metabolic activity, assessed by MTT test, of the cells grown on 160−, 280+, and 2400+ titanium was significantly decreased against control.However, the tested materials did not differ among each other, which indicated that all tested materials were cytocompatible.This is in line with the numerous studies demonstrating the biocompatible character of titanium as a substrate for cell culturing [40][41][42][43].
The usage of a second cell model (hFOB 1.19) revealed that one of the tested materials seems to be as good as control with respect to metabolic activity of the osteoblasts cultured on its surface.It was the material with the smallest grain size that seemed to be consistent with the studies that detected that the smaller the grain size, the better the viability [44][45][46].Other studied materials were significantly worse than control and 160+.Interestingly, this result was reached only for one of two materials with one certain grain size.This observation indicated that two different sections differ in the viability of cells grown on its surface, which is in agreement with the study of Hoseini et al., who conclude that crystallographic texture, rather than grain size, plays a principal role in the surface biocompatibility [47].
It is well established that the proteins of extracellular matrix, membrane receptors, and cytoskeletal proteins are responsible for cell-substrate interactions.That is why we decided to analyse two important cytoskeletal proteins actin and vinculin by immunocytochemical staining.Actin is a critical player in many cellular functions, such as cell motility and the maintenance of cell shape and polarity [48].Vinculin is a cytoplasmic actin-binding protein enriched in focal adhesions and adherens junctions required for strong cell adhesion [49].As early as 2 h after seeding, the cells adhered and began to spread (Figure 7(a)).The cells displayed wellspreading morphology with many vinculin spots after 24 h (Figure 7(b)).This observation proved that the adhesion phase occurred on all tested materials.
We also intend to examine differences between two used cell models.Fibroblasts represent soft tissue and osteoblast hard tissue, and the dental implant needs to be in contact with both.We did not record significant differences in viability among tested materials plating with fibroblast cells.When we used osteoblasts as a cell model, we recorded that material with a grain size 160 nm with longitudinal section seemed to be as good as a conventional culture plate with respect to cell viability and proliferation.Therefore, this material could be recommended for a detailed study of cell behaviour in vitro as well as in vivo.

Conclusions
The aim of this study was to evaluate if any of the six studied materials is better than others with respect to biocompatibility and cell proliferation.Similar cellular behaviour was observed on all studied biomaterials.There were differences related to the initial phase of attachment, but not in proliferation.Furthermore, the results reported in this paper indicate that osteoblasts grow on material with a grain size of 160 nm with longitudinal section as well as on a conventional culture plate, whereas, for other studied materials, we observed decreased viability.This material could be recommended for further evaluation with respect to osseointegration in vivo.

Figure 1 :
Figure 1: Photographs illustrating nanostructured titanium samples.(a) Macroscopic image; (b) details of the surface; (c) SEM images of longitudinal section; (d) SEM images of transversal cross-section.

Figure 2 :
Figure 2: SEM photographs of the sample surfaces with different grain sizes and sections.(a, b, c) SEM images of longitudinal section with grain sizes of 160, 280, and 2400 nm, respectively; (d, e, f) SEM images of transversal cross-section with grain sizes 160, 280, and 2400 nm, respectively.

Figure 3 :
Figure 3: The surface roughness expressed by arithmetical mean roughness Ra and ten-point mean roughness Rz.The standard errors were calculated from three independent measurements.Error bars indicate means ± standard deviations.

Figure 4 :Figure 5 :
Figure 4: The area of surface (percentage) of six studied titanium materials with different grain sizes and sections occupied by human fibroblasts HFL1 at 6 h and 24 h after plating.Results from two distinct experiments on the basis of duplicate determination were combined.Error bars indicate means ± standard deviations.

Table 1 :
Medians of viability of two cell lines (hFOB 1.19, HFL1) estimated by MTT assay expressed as % of positive control.