Assessment of Biomimetic Modification on a Novel Low Elastic Modulus Ti–Nb–Hf Alloy for Dental Implants

A simple review of research literature and Research and Development trends (R&D) in the field of dental implants shows an exponential increase in their quantity. The impressive growth in the contributions of nanoscience, in general and nanotechnology/nanomaterials applied to dental care, in particular, also shows the priorities investments and substantive effort of the global research community in this area. Nevertheless, there are still many research challenges and huge opportunities to optimize dental implants. The essential purpose of this study is to assess biomimetic modifications on a novel low elastic modulus TiHfNb Alloy Ni-free. For the mimic surface samples were initially bioactive with Oxygen Plasma (OP) or Piranha etching (Pi). Later three different silanes were used (CPTES, GPTES or APTES+CL) and finally RGD or mixtures 1:1 of RGD:FHRRIKA or RGD:PHSRN were immobilized on the surface. To evaluate the biomimetic modification, cells adhesion studies were performed. For the chemical characterization different techniques were used, including, inter alia: X-ray Photoelectron Spectroscopy (XPS), Zinc-substitution complex technique (ZC), and immunofluorescence. Experimental results showed that the best activation method was the OP, and the biomimetic, modification is a well-grounded research strategy to improve the cell adhesion. *Corresponding author: Paredes V, Biomaterials, Biomechanics and Tissue Engineering Group (BIBITE), Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), Barcelona, Spain, Tel: 34934010717; E-mail: virgiparedes@gmail.com Received October 07, 2015; Accepted October 20, 2015; Published October 29, 2015 Citation: Paredes V, Salvagni E, Manero JM (2015) Assessment of Biomimetic Modification on a Novel Low Elastic Modulus Ti–Nb–Hf Alloy for Dental Implants. J Material Sci Eng 4: 193. doi:10.4172/2169-0022.1000193 Copyright: © 2015 Paredes V, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. a shape memory effect and superelasticity [12], though Ni ions released produce adverse reactions (allergy) [13]. Thereby, some metallic materials could be considered well tolerated, however, these materials are not inert, and consequently Ni-free, Alfree and V-free alloys are part of the current trend to develop alloys able to overcome the incompatibility of providing a biological balance [11]. Different authors have reported that Ti alloy containing elements no toxic as Nb, Zr, Ta, Mo, Hf, Fe, Sn, Zr; and β-Ti phase, improve mechanical properties, low elastic modulus, biocorrosion resistance, also they are biocompatible and have not allergic problems [1, 4-7]. Once selected the material, another important parameter to considerer is the biological response, Thus, the future dental implant should be developed understanding the interaction metal-tissues through the evaluation of the relation between implant, proteins and cells. How to improve the interaction between material and cells? The stabilization of the bone–implant interface can be achieved by the adhesion of a series of proteins, because inside the body we have different sequences of peptide called proteins. For this purpose we have to know what kind of protein we will use. Osteoblast adhesion takes place by different mechanisms: the most investigated implies the interaction with RGD sequences (Arg-Gly-Asp), because this can Journal of Material Sciences & Engineering J o u r n a l o f M ate rial Sciences & Egin e e r i n g ISSN: 2169-0022 Citation: Paredes V, Salvagni E, Manero JM (2015) Assessment of Biomimetic Modification on a Novel Low Elastic Modulus Ti–Nb–Hf Alloy for Dental Implants. J Material Sci Eng 4: 193. doi:10.4172/2169-0022.1000193


Introduction
The implants have a purpose to replace a part or a function of the body in a manner that will be safe and physiologically acceptable. However, the material to replace teeth, bones or joints are complex, because the selection of materials depend of different ideals factors, such as biocompatibility, excellent corrosion resistance, high mechanical strength, good wear resistance and low elastic modulus [1]. Inequality of Young´s moduli has been identified as one of the main causes for implant loosening followed by stress shielding [2].
Currently, the material most commonly used as implants to bone regeneration, have elastic modulus between 220 GPa (CoCr) and 110 GPa (Ti), however the cortical bone has an elastic modulus of 7-25 GPa [3,4], then, there is a tendency to show a stress shielding effect [5][6][7]. Therefore, low modulus alloy are nowadays desired, because the modulus required for implant must be more similar to that of natural bone, which will inhibit bone atrophy and induce good bone remodeling as stated above [4][5][6].
The number of dental implant has increased in the past decades, reaching about one million per year. Their success are related to the osseointegration rate, which depends of their chemical composition, geometry and topography [8,9]. Moreover, the dental implants interact with physiological fluids, containing biologically ion species able to affect the surface, the environment and the biological system [10].
Between the dental metallic materials for implants, the Ti alloys have favorable specific strength (strength to density ratio) and lowest elasticity´s modulus [11]. For that reason the Ti-6Al-4V and NiTi were of the first Ti biomaterials used in implantable devices. However, some studies have shown that either Al or V ions are able to cause long-term health problems, such as: reaction with tissue in animals, neurological disorders, Alzheimer´s disease, and also this alloy have elastic modulus higher than bone [1]. Also NiTi (Nitinol) has been used as implant due be recognized in a wide range of cells. Secchi indicate that RGD on Ti stimulates cell differentiation and confers to the surface a resistance property to apoptosis (cell death) [14].
Recent studies have concluded that a mixture of RGD with other sequences improves the cell response [15][16][17][18]. Rezania worked with RGD and FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala). Their results showed that by using short peptide sequences with heparin-adhesive motifs is possible to improve the cell interaction and affect the longterm formation of mineralized ECM [19]. Also different author had indicated that FHRRIKA add specific advantage for substrates and promote the osteoblast adhesion [15,[20][21][22][23].
Researchers such as Benoit, uncovered that PHSRN improve the adhesion and proliferation of osteoblast, and also contribute with the production of phosphaphate alkaline, which is an indicator of osteoblastic function [24]. Other studies indicate that mixtures RGD and PHRSN mimic the microenvironment of the cells. Consequently this information provides opportunities for developing biomaterials [15].
Therefore in this study a Ti (TiHfNb) Ni free alloy was used. This shows good resistance and presents an elastic modulus of 74 GPa [12,13,[25][26][27][28][29]. Although, excellent qualities of this material lacks bioactivity. To overcome this drawback different biomolecules were incorporate. Namely CGGRGD (Cys-Gly-Gly-Arg-Gly-Asp), and mixtures 1:1 CGGRGD: CGGFHRRIKA (Cys-Gly-Gly-Phe-His-Arg-Arg-Ile-Lys-Ala) and CGGRGD: CGGPHSRN (Cys-Gly-Gly-Pro-His-Ser-Arg-Asn) [30,31]. The Cys sequence was used due to the fact that this has a Sulphur atom in its lateral chain that this element could be detected by X-ray Photoelectron Spectroscopy (XPS) and is highly reactive. Finally, cells adhesion studies on modified surfaces were made to evaluate the biomimetic modification.

Materials
The Ti alloy (TiHfNb) was fabricated by vacuum arc melting into UPC laboratory using high purity metals 99,99% (Table 1). Thus the buttons were homogenized at 1100°C for to 12 h, treated for 1.5 h at the same temperature, and finally quenched in ice water.
The buttons were cut of 8mm × 5 mm and 2 mm of thickness. The samples were polished using SiC abrasive papers (600 to 1200) and finally were used napped polishing pad with colloidal alumina (1 and 0.05 µm). The samples were degreased by ultrasonication with acetone, water and ethanol, and dried with compressed air.

Biomimetic modification
That objective can be realized through the biomimetic modification of substrate. When exposed to the air the metals are oxidized. Thus they have an oxide layer with impurities that are not convenient. Therefore it is necessary to do the activation, in order to obtain the cleaning and activation of the surface. Once the surface is clean and active it is necessary to make the silanization, followed of the adhesion of peptide. In order to know which kind of sequence is the best and at the same time to show that biomimetic process produces a good cell response studies of cell adhesion were make.
Activation process: Smooth Ti alloys samples were bioactivated with i) Oxygen Plasma (OP) or ii) Piranha (Pi), to remove the impurities of carbon and to generate highly active surface through the addition of OHgroups [32,33]. Based on the highest concentration of the OHgroups and the removal of carbon the best treatment was selected.
Depending on the two higher atomic percentage of Si the two silanes were selected for the next step of biomolecule immobilization.

Immobilization of peptide sequences:
The best silanization samples were coated with different peptides as CGGRGDS, CGGPHSRN, CGGFHRRIKA, or mixtures of them (1:1): CGGRGDS + CGGPHSRN or CGGRGDS + CGGFHRRIKA. Non-treated Ti alloys samples were used as control [15,19,22,[38][39][40][41]. For the immobilization of the peptide sequences, the silanization samples were incubated overnight in phosphate buffer solution (PBS) containing one peptide or one mixture. Finally samples were extracted from PBS solution and stored in a desiccator.

Cell response
For the cell adhesion assays, were used a mesenchymal cell of rat to induce the osteoblast response. Prior studies of viability show that the optimal time adhesion is to 6 h in serum free media. For the seeding of cells samples were treated with 1% bovine serum albumin solution (PBS-BSA (1%)) for 30 min in order to inhibit the unspecific protein binding, Thus the sterilization was realized immersing samples in ethanol for 10 min and finally samples were washed with sterile PBS. The best cell response will depend on cell number.

Surface characterization
Surfaces were characterized by X-ray Photoelectron Spectroscopy (XPS), and Zinc-complex Substitution Technique (ZT). The in vitro effect of modified surfaces was evaluated by immunofluorescence. Mann-Whitney and Kruskal-Wallis statistical tests were used (p-value<0.05).

X-ray Photoelectron Spectroscopy (XPS):
The ESCA 5701 (Physical Electronics, PHI 10) with monochromatic Mg Kα X-ray source (300.0 W, E = 1,253.6 eV) was used for the measurements. Survey spectra were collected with pass energy of 190 eV. High resolution (HR) spectra were collected with pass energy of 24.95 eV. The binding energies were corrected based on C 1s peak at 284.8 Ev [42,43]. Multipak spectrum data analysis software was used to do curve-fitting of the spectra and to calculate component composition from the peak areas. Such analyses are accurate to ±10 % [44], also the binding energies reported have an error in the range of ±0.1 eV [43].

Zinc-complex Substitution Technique (ZT):
This is an experimental technique reported in the literature [32,33], which consists of immersing the samples in a mixture 2:1 of ammonium chloride solution (4.0M) and zinc chloride solution (0.4M). During immersion, one zinc complex is formed with two hydroxyl groups,  Cell response: For each of the adhesion assays 6×10 3 cells were seed per well of 0.95 cm 2 . The cell adhesion process was characterized by i) immunofluorescence through the staining of cell nuclei with DAPI (4,6-diamino-2fenol-dihydrochloride) the quantification was performed through Image J software. For this characterization was necessary to do the fixation and dehydration of cells.

Activation process
The nomenclature used is i) Control (Ti alloy untreated), ii) TiOP (Ti alloy+OP) and iii) TiPi (Ti alloy + Pi). Scheuerlein reported that to accept or reject a cleaning treatment a cleanliness criterion was based on the maximum allowable atomic carbon surface concentration [45]. Thus, in terms of cleanliness, the XPS allowed to know the chemical composition in atomic percentage. Through Figure 1 it is possible to see the behavior of carbon and oxygen for each sample, where the control sample have a higher carbon atomic concentration (42%), and a decrease of carbon for the oxidation samples (11% for OP, and 32% for Pi).
Xia reported that an atomic carbon surface concentration of 18% is typical for organic contaminants levels on clean oxide surface [46]. Therefore, it is possible confirm due to the fact that samples with OP had a lower amount of carbon, thus those have been cleanliness.
Thereby this treatment cleans better the surfaces than Pi treatment.
Regarding OHgroup on the surface Figure 1 allows observing an increase of oxygen in both oxidation samples. Thus it is necessary to know if: will there be OHgroups on the surface?
In order to know if there are OHgroup on the surface the XPS high resolution of O 1s spectrum was used. Results of Table 2 and Figure 2 agree with reported by other authors in previous studies, which show that there are at least 3 peaks or 3 different kinds of oxygen species: oxide (O 2-) , hydroxide or hydroxyl group (OH -) and absorbed water (H 2 O) [32,33,[46][47][48][49].
Thus, through the O 2it is possible to confirm that Pi treatment is able to increase the oxide layer more than OP. However, in terms of OHgroups, both methods were efficient, though the OP had a highest percentage.
Moreover, for efficient surface activation process, some authors have reported that it is convenient an increase in the ratio between the hydroxyl groups of the activated surface and the metal oxides (OH -/ O 2-), because it indicates that has occurred an increase in OHand a decrease of O 2- [32,33,48]. For that reason another important point is to calculate the ratio between OH -/O 2and choose the higher value as the best [33,49,50]. Thereby the Oxygen Plasma resulted to be the better activation method in terms to introduce a higher amount of OHgroup per percentage of oxide (OH -/O 2-).
By XPS High resolution it is possible to know the percentages of chemical composition of OHgroup on the surface and inside of oxide layer (about 10 nm) [51]. Thus, there are authors, who established differences between hydroxyl groups on the surface and "active hydroxyl groups"(OH-act). These last react easily and are on the outer layer [32,33]. In order to distinguish hydroxyl groups active on the outer layer, an alternative technique (Zinc-complex substitution) was used. Figure 3 shows the concentration of active hydroxyl groups (C OH-act ) per unit area on the outer layer of active samples. The results confirm that both activation methods are able to introduce OHgroup on the surface. However, over again, the Oxygen Plasma had the best results in terms of C OH-act .

Silanization process
In this phase were used the samples active with OP, thus the nomenclature used is i) TiC (Ti alloy +OP+CPTES TiC), ii) TiG (Ti alloy+OP+ GPTES) and iii) TiAC (Ti alloy+OP+ APTES+CL). Before to analyze the silanized samples is convenient to know the chemical composition of each silane used (Figure 4).
Fraiol reported that the presence of silanes on the surface, can be      detected by the appearance of the peak of silicon (Si 2p) on the surface by XPS [52]. Also different studies indicate that the peak Si 2p emerges on silanized samples because these have bonds of Si-O-metal and O-Si-O (siloxane) [53][54][55][56]. Table 3 shows all silanized samples have silicon on the surface. In particular the TiAC has greatest amount of Silicone (12% of Si 1s). This is a lowest amount of metallic elements (0%), for that it seems that TiAC have a thicker layer. This is followed by TiC samples which show a higher amount of Silicon than TiG (5% and 2% respectively). Otherwise the existence of Cl (2% Cl 2p) to the TiC confirms the presence of this silane, because CPTES has a Cl atom in their lateral chain.

Control
Also an increase of C 1s in all silanized samples, because each silane contains carbon chains. Therefor the higher amount corresponds to TiAC (55%), due to the fact that it has longer carbon chains. Alike the TiAC showed major percentage of N 1s (10% of N 1s compared to 2% for untreated sample) due to the chemical composition of silane.
In sum, depending on the XPS information it can be inferred that sample TiAC generates a better result, because: i) TiAC contains as many Si 2p (12%) than TiC (5%) and TiG (2%), and ii) TiAC has a lower percentage of metallic elements, due to the fact that it has a thicker layer because it has a longer chain. Therefore the immobilization of peptide sequences will be used TiC and TiAC.

Immobilization of peptide sequences
How know, if there is a binding of the peptide. The strategy used peptide sequences with Cysteine, because this could be detected by XPS trough of S 2p, considering that the Cys has a thiol (-SH) in its lateral chain.
By XPS it was possible to identify all samples with biomolecules by the presence of S 2p, which indicate the existence of peptide sequence on the surface ( Table 4). Regarding of C 1s, the results of TiC samples show an increase to the surface with mixture, because to the presence on dipeptide and carboxyl groups.
However, no statistically significant differences occurred between samples TiAC+peptide and TiC+peptide, although it has been found that TiAC have higher atomic percentage of silicon surface. Thus, the assessment the biomimetic modification will be depending on which peptide sequence improve the cell response.

Cell response
In order to evaluate the cell adhesion process it was used the immunofluorescence to staining the nuclei, actin filament and focal points. The experimental results are showed in Figure 5, where is possible to observe that higher amount if cell are present on samples with APTES+CL (TiAC).
For quantification of cell adhesion process, a study was conducted by staining of cell nuclei with DAPI and the results are shown in Figure  6. Depending on the results obtained it can be summarized in terms of the density of cells, the biomimetic modification with APTES +CL generates better results.

Conclusions
This research demonstrated that by Oxygen Plasma it is possible to obtain a best removal of carbon from contamination. Furthermore,