Preparation of Laser-Modified Ti-15Mo Surfaces With Multiphase Calcium Phosphate Coatings

10, 2020 Multiphasic bioceramic scaffolds has been enhanced for dental and orthopedic applications. In this perspective, the laser surface texturing of metallic surfaces combined to bioactive calcium phosphate coatings have shown to be promising and economically feasible for biomaterial clinical applications. Ti-15Mo alloy samples were irradiated by pulsed Yb: YAG laser beam. The formation of HA and other calcium phosphates phases by biomimetic method should occur in the presence of Ca 2+ , PO 43- , Mg 2+ , HCO 3- , K + and Na + . The modified surfaces were submitted to thermal treatment at 380 and 580°C. The results showed the processes of fusion and fast solidification from the laser beam irradiation, inducing the formation of stoichiometric α-Ti, TiO 2 and non-stoichiometric titanium oxides, Ti 3 O and Ti 6 O with different oxide percentages depending on applied fluency (fluency of 0.023, 0.033, 0.040 and 0.048 J/mm 2 ). The morphological and physicochemical properties have indicated the formation of a multiphase bioceramic coatings. It was observed the formation of amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), and magnesium phosphate (Mg 3 (PO 4 ) 2 ) phases at 380°C, whereas β-TCP (tricalcium phosphate), OCP, and substituted β-TCP with Ca 2,589 Mg 0,41 (PO 4 ) 2 were obtained at 580°C. Therefore, the multiphasic bioceramic modified Ti-15Mo surface could enhance osteointegration for


Introduction
Calcium phosphates has been used in a variety of applications for the treatment of the bone system, since insulated material that surface coating of metallic implants [1][2][3][4][5][6][7] .
The biomimetic coating method is based on the heterogeneous precipitation on titanium substrates and their alloys. The nucleation and growth of the calcium phosphate coating occurs after immersion in a balanced salt solution (Hank's solution or SBF) at 37°C for several days 4,16 . This process is similar to the process of bone biomineralization 4,17,18 . The modified biomimetic method represented a major advance in the area of biomaterials. The growing interest in the use of other phases of calcium phosphates has resulted in more promising properties than the HA phase, new strategies have been described in bioceramic coating on metal surfaces 16,19 .
In our previous work 2 , Ti-Mo laser-activated surfaces were coated by sol-gel calcium phosphates, indicating a mixture of phases under diferents temperature control. As a continuation of our previous work, this study has evaluated six different simulated body fluid solutions, which were called modified SBF, in order to design a multiphase bioceramic coatings with controlled chemical deposition on metallic surface. The aim of this work was to evaluate the morphological and physicochemical properties of the surfaces of the Ti-15Mo alloy modified by Yb: YAG laser beam, as well as deposition of bioactive ceramics using the modified biomimetic method.

Laser-activated surface modification
Samples of Ti-15Mo alloy (4 mm length, 4 mm wide, 2mm thick) were submitted to Yb:YAG multipulse laser irradiation using a Laser OmniMark 20 F (OmniTek, São *e-mail: carla.riccardi@unesp.br Paulo, Brazil) (λ = 1090 nm) at a short exposition time (1 minute). Our research group has evaluated the topography of metallic surfaces in order to relate parameters of Ti-15Mo surface, such as morphology and roughness and surface energy, depend on the formed phases 2,3 . The surfaces were modified under ambient pressure and air, using the parameters (power, frequency and scan speed) with four fluency (ablation) of 0.023, 0.033, 0.040 and 0.048 J/mm 2 (n= 5 for each treatment), respectively (Table 1). After irradiation, the samples were treated ultrasonically and separately in solutions of ethyl alcohol, acetone and deionized water, followed by oven-drying and characterization.

Preparation of the modified SBF solution and biomimetic coating
The irradiated samples were immersed in modified SBF solution (SBFMg). This solution contained different ions in order to improve the formation of the phases of interest. The reagents used were: NaCl, K 2 HPO 4 , CaCl 2 .2H 2 O, MgCl 2 .2H 2 O and HCl supplied by J. T. Baker; Tris (hydroxymethyl) aminomethane was purchased from Mallinckrodt. Table 2 indicates the ionic concentrations of the SBFMg solution used to obtain the calcium phosphate coatings on the laser beam irradiated Ti-15Mo surfaces. The preparation of the SBFMg solution was modified from reported protocol by Aparecida (2007), in order to minimize the possibility of solution loss caused by its precipitation.
The substrates were washed sequentially with alcohol, acetone and deionized water. To obtain the calcium phosphate coatings using the SBFMg solution, all the samples were immersed in 50 mL of modified SBF solution (pH 7.4), and remained in controlled temperature condition at 37ºC for 4 days 11,16,20 . The solution was exchanged every 24 hours for the purpose of promoting the super-saturation conditions of the solution and, consequently, inducing the formation of the calcium phosphate coating. After the period to obtain the coatings, the samples were air dried and submitted to thermal treatment at 380 and 580ºC for 3 hours, without atmospheric control. The heating and cooling rate used was 5°C/minute.

Characterization
All the coated and uncoated samples were characterized by scanning electron microscopy (SEM), using a Zeiss EVO LS-15, with EDS/EBDS Oxford INCA Energy 250 system. The X-ray diffraction analysis was performed in a Siemens D5000 X-ray diffractometer, using a scan angle of 5 at 60º with a step size of 0.02 (2θ). Each sample was subjected to a counting time of 10s/step in a Bragg-Brentano configuration, using Cu (kα1) radiation. Quantification by Rietveld refinement was performed in a Rigaku RINT-2000 X-ray diffractometer with rotating anode, operating under the experimental conditions of 42KV, 120mA, with divergence slits, scattering angle of 0.5º, 5 mm horizontal opening of the divergence slit, 0.3 mm receiving signal, 5° Soller, copper anode, and wavelengths of Kα 1 = 1.55056 Å and kα 2 = 1.5444 Å, Iα 2 /Iα 1 = 0.5. The chemical bonds of the calcium phosphates coatings were characterized by vibrational infrared spectroscopy, using a Bruker Vertex 70 FTIR spectrophotometer equipped with a diffuse reflection DRIFT Collector TM .

Morphological properties
The micrographies of the surfaces of the uncoated samples Ti-15Mo alloy submitted to laser beam using different fluencies (0.023, 0.033, 0.040 and 0.048 J/mm 2 , respectively) are presented in Figure 1A. It can be observed the increased fluency, due to longer exposure time of the laser beam to the alloy surface, promotes typical morphologies with different surface energies. This can be explained through the formation of new structures (metal oxides) produced during the fast melt and solidification process 2,3,21 Figure 1B shows the morphologies of the coatings obtained in samples 1, 2, 3 and 4, using the SBFMg solution and heat treated at 380ºC. It was possible to identify the formation of a coating with morphology characteristic of the ACP 2, OCP and Mg 3 (PO 4 ) 2 phases 2,6,10, 12,13,17,18,22 .
The morphologies of the coatings obtained for samples 1, 2, 3 and 4, using the SBFMg solution and heat treatment treated 580ºC are presented in Figure 1C. The formation of a multiphase coating was evidenced, evidenced by the presence of particles with different morphologies and size, characteristic of the phases of β-TCP, TCP replaced with magnesium -Ca 2,589 Mg 0,41 (PO 4 Figure 2 shows the diffractograms of samples (0.023, 0.033, 0.040 and 0.048 J/mm 2 ), respectively.

XRD Rietveld refinement
It was possible to produce the formation of stoichiometric and non-stoichiometric oxides as predicted by the fluency equation. X-ray diffraction spectra revealed (Figure 2A Table 3 shows the oxide phases percentage obtained by Rietveld refinement, corresponding to laser beam-irradiated Ti-15Mo surfaces 30,31 . It can be observed the fusion and solidification process under ambient air, inducing the   formation of titanium oxides with different degrees of oxidation by laser ablation. The oxidation mechanism of titanium is complex owing to the high solubility of oxygen in the hexagonal-close-packed (h.c.p.) structure of α-titanium.
A recent study has shown there are two other potential intersticial site (hexahedral and crowdion) in α -Ti where the oxygen can be located 32 . The presence of the Ti 3 O and Ti 6 O , substoichiometric phases can be explained by interstitial oxygen diffusion in the Ti lattice 33 . The X-ray diffraction patterns of the bioceramic coatings, obtained using the SBFMg solution on the surfaces of the samples (1: F = 0.023 J / mm 2 , 2: 0.033 J / mm 2 , 3: 0.040 J / mm 2 and 4: 0.048 J / mm 2 ), Figure 2B. All peaks corresponding to the Ti-15Mo alloy (#: 89-4913) were identified, formation of an ACP 2 phase mixture OCP (#: 26-1056) and magnesium phosphate (#: 48-1167) 29 .
The formation of the ACP phase to the HA phase can occur directly from ACP1, whereas its transformation through the formation of intermediates occurs with ACP2 as another intermediate 2,27,34 .
The use of the SBFMg solution favors the formation of OCP (octacalcium phosphate) due to the presence of the Mg 2+ ion which allowed the crystallization of the ACP 2 and its partial transformation to OCP and the appearance of the magnesium phosphate phase. It was observed the amount of Mg 2+ incorporated into calcified tissues associated with the calcium phosphates phase decreases with stronger calcification, leading to changes of the bone matrix that determines the bone fragility 13,35 . Therefore Mg 2+ ions were incorporated into calcium phosphate ceramics, it is expected the in vivo process of this synthetic materials is more similar to bone mineral, as compared to Mg free synthetic materials 13,36 Figure 2C shows the X-ray diffraction patterns of the bioceramic coatings, obtained using the SBFMg solution on the surfaces of samples 1, 2, 3 and 4. In all samples the peaks corresponding to the Ti-15 Mo (#: 89-4913), the formation of a mixture of phases tricalcium phosphate (β-TCP) (#:70-2065) TCP replaced with magnesium -Ca 2,589 Mg 0,411 (PO 4 ) 2 (#:87-1582), magnesium phosphate -Mg 3 (PO 4 ) 2 (#: 48-1167) e OCP (#: 26-1052) 29 . The formation process of the β-TCP and Ca x Mgy(PO 4 ) 2 phases may be related to the decomposition of the non-stoichiometric hydroxyapatite phase, between 600 and 750°C, reaction below 13,27,37,38 : The Mg 2+ ion is one of the most abundant trace ions in biological hard tissues. In dental enamel, the concentration is 0.4%, in the 1% dentin and in the bone 0.5%. The amount of Mg 2+ in dental enamel increases from the surface to the enamel / dentin junction area. The properties of calcium phosphates of biological interest can be affected by the presence of Mg 2+ . This ion has been reported as responsible for the calcium phosphate crystallization disorder, especially HA, when present in the solution in quantities sufficient to compete with Ca 2+ ions. Studies have shown that when the Mg / Ca molar ratio of the solution is greater than 0.05, formation of Mg 2+substituted TCP will occur 13,37-39 .

Fourier transform infrared spectroscopy
The spectra in the middle infrared region of the bioceramic coatings using the SBFMg solution on the surfaces of samples 1, 2, 3 and 4 are shown in Figure 3A 380 °C and 3B 580 °C.
In the Figure 3B, it can be observed that all the spectra present bands in the regions between 1129-946 and 730 cm -1 which indicate the asymmetric stretching of the P-O-P bond, and a band in the 1242 cm -1 region relative to the P = O stretch 40,41 . For samples (1, 2, 3 and 4), the bands 630 and 572 cm -1 were associated with the stretching of the OH-group, the vibration of the PO 4 3group and the depletion of the PO 4 3group 42 . The band at 1646cm -1 is due to the incorporation of water molecules. The bands at 1370 and 1474 cm -1 may be associated with the vibration of the CO 3 2group, from the CO 2 of the atmosphere during the processes of dissolution, agitation, reaction and calcination, or to the formation of carbonated hydroxyapatite due to the possibility of substitutions occurring of the ions PO 4 3or hydroxyl of the hydroxyapatite by the ion CO 3 27,41,43,44 .
For the coatings using modified SBFMg at 580°C (Figure 3B), all the spectra bands in the regions between 1240-940 and 760-720 cm -1 which indicate the asymmetric stretching of the P-O-P bond, and a band in the 1240 cm -1 region relative to the stretching of P = O 40, 41 . In all samples (1, 2, 3 and 4) the presence of the 630 and 545 cm -1 regions was observed, which may be associated with the OHgroup stretching, the PO 4 3group vibration and the unfolding of the group PO 4 3and refer to the probable formation of the hydroxyapatite phase 2,42,45 . Bands in the region of 1733-1630 cm -1 are attributed to the incorporation of water molecules. The bands 1386-1455 cm -1 may be associated with the CO 3 2-, vibration from the CO 2 of the atmosphere during the processes of dissolution, agitation, reaction and calcination 24,27,41,43,44 ,

Conclusion
Bioceramics coatings have been deposited on metal and its alloy surfaces by laser ablation process. Multiphasic calcium phosphates must exhibit a combination of enhanced bioactivity and mechanical stability that is difficult to obtain in single-phase materials. In the present study, it has been also demonstrated the formation of different stoichiometric and non-stoichiometric oxides, such as α-Ti, TiO 2 , Ti 3 O, Ti 6 O, as well as the different oxide percentages depending on the applied fluency. This aspect has provide typical morphologies of the calcium phosphates phases. In this perspective, a multiphase bioceramic coatings on Ti-15Mo surfaces could be obtained depending on the thermal tratment performed to 380 and 580°C. Therefore, the multiphase bioceramic coatings deposited on Ti-15Mo surfaces can be further improved by providing an biocompatible and long term performance for biomedical applications, including bone regeneration in orthopaedics, oral and maxillofacial surgery.

Acknowledgments
This work has been supported by CNPq -The Brazilian Council for Research and Scientific Development.