Osteoblast biocompatibility and inhibition of bacterial adhesion to thermally and chemically treated TiAlV alloy

1. INTRODUCTION

Material-tissue interface and the long-term success or failure of the integration of an implant in the body are closely correlated with the properties of the implant devices and specifically with their surface properties, such as wettability, texture or chemical composition. The TiAlV alloy has an excellent in vivo performance, which makes it the most commonly used prosthetic metallic materials for rods in prostheses for joint and dental implants. However, new research into this alloy, based on the changes in chemical composition, has been carried out with the aim of obtaining alloys with optimal microstructures designed to achieve mechanical properties similar to those of bone and with high corrosion resistance and enhanced biocompatibility promoted by faster integration with bone tissue and minimum bacterial proliferation (Mareci et al., 2009Mareci, D., Lucero, V., Mirza, J. (2009). Effect of replacement of vanadium by iron on the electrochemical behaviour of titanium alloys in simulated physiological media. Rev. Metal. 45 (1), 32-41. https://doi.org/10.3989/revmetalm.0750.).

The addition of elements in minor contents can determine the surface performance of Ti-based alloys. The vanadium content in this alloy cause toxicity over time, when interacting with biological systems as some studies have reported (Siqueira et al., 2009Siqueira, R.P., Sandim, H.R.Z., Hayama, A.O.F., Henriques, V.A.R. (2009). Microstructural evolution during sintering of the blended elemental Ti-5Al-2.5Fe alloy. J. Alloys Compd. 476 (1-2), 130-137. https://doi.org/10.1016/j.jallcom.2008.09.004.). So research has been addressed to produce new alloys with a Ti base where the V has been substituted by other elements, such as Fe, which also stabilizes the β phase in the alloy (Simsek and Ozyurek, 2019Simsek, I., Ozyurek, D. (2019). Investigation of the wear and corrosion behaviors of Ti5Al2.5Fe and Ti6Al4V alloys produced by mechanical alloying method in simulated body fluid environment. Mater. Sci. Eng. C 94, 357-363. https://doi.org/10.1016/j.msec.2018.09.047.). Other alloys that have been under investigation using Niobium and Iron instead of Vanadium are, for example, Ti6Al7Nb and Ti5Al2.5Fe (α+β) phase (Niinomi, 2002Niinomi, M. (2002). Recent metallic materials for biomedical applications. Metall. Mater. Trans. A 33, 477. https://doi.org/10.1007/s11661-002-0109-2.; Jackson and Ahmed, 2007Jackson, M.J., Ahmed W. (2007). Titanium and Titanium Alloy Applications in Medicine. In Surface Engineered Surgical Tools and Medical Devices. Springer US, Boston, pp. 533-576. https://doi.org/10.1007/978-0-387-27028-9_15.). Research of the wear and corrosion behavior of Ti5Al2.5Fe and Ti6Al4V alloys processed by mechanical alloying in a simulated body environment show that although there is an improvement in the wear resistance, both alloys show pitting corrosion in simulated fluids at body temperature (Simsek and Ozyurek, 2019Simsek, I., Ozyurek, D. (2019). Investigation of the wear and corrosion behaviors of Ti5Al2.5Fe and Ti6Al4V alloys produced by mechanical alloying method in simulated body fluid environment. Mater. Sci. Eng. C 94, 357-363. https://doi.org/10.1016/j.msec.2018.09.047.). Alloying elements such as Al, V, and Nb in titanium alloys may be incorporated into the thin oxide film and that probably influences the adsorption of proteins and their accommodation on the surface, modifying the surface-cell interaction (Jenko et al., 2018Jenko, M., Gorenšek, M., Godec, M., Hodnik, M., Batič, B.Š., Donik, Č.J., Grant, T., Dolinar, D. (2018). Surface chemistry and microstructure of metallic biomaterials for hip and knee endoprostheses. Appl. Surf. Sci. 427, 584-593. https://doi.org/10.1016/j.apsusc.2017.08.007.). Other surface property, which has an effect on the interaction with proteins is the isoelectric point of the surface which can change depending on the elements that are alloyed (Sittig et al., 1999Sittig, C., Textor, M., Spencer, N.D., Wieland, M., Vallotton, P.H. (1999). Surface characterization. J. Mater. Sci. Mater. Med. 10, 35-46. https://doi.org/10.1023/A:1008840026907.).

On the other hand, the modification of the surfaces of these alloys are also performed in order to improve their properties of biocompatibility and osseointegration and their resistance to bacterial colonization. It is known that Staphylococcus species is responsible for more than 60% of prosthetic infections (Zorn et al., 2011Zorn, G., Baio, J.E., Weidner, T., Migonney, V., Castner, D.G. (2011). Characterization of Poly(sodium styrene sulfonate). Thin Films Grafted from Functionalized Titanium Surfaces. Langmuir 27, 13104-13112. https://doi.org/10.1021/la201918y.). Bacteria may also be the cause of the orthopedic implant removal following aseptic loosening (Geetha et al., 2009Geetha, M., Singh, A.K., Asokamani, R., Gogia, A.K. (2009). Ti based biomaterials, the ultimate choice for orthopaedic implants - A review. Prog. Mater. Sci. 54, 397-425. https://doi.org/10.1016/j.pmatsci.2008.06.004.; Raphel et al., 2016Raphel, J., Holodniy, M., Goodman, S.B., Heilshorn, S.C. (2016). Multifunctional coatings to simultaneously promote osseointegration and prevent infection of orthopaedic implants. Biomaterials 84, 301-314. https://doi.org/10.1016/j.biomaterials.2016.01.016.). Specifically, Staphylococcus epidermis form biofilms on the prostheses surface, being the principal causes of antibiotic resistance and responsible for the implant replacement (Mareci et al., 2009Mareci, D., Lucero, V., Mirza, J. (2009). Effect of replacement of vanadium by iron on the electrochemical behaviour of titanium alloys in simulated physiological media. Rev. Metal. 45 (1), 32-41. https://doi.org/10.3989/revmetalm.0750.). So, the success of the implant is based on promoting osseointegration and inhibiting microbial adhesion.

Bearing in mind the importance of this, new surface modification procedures are being developed that do not impair the mechanical properties of the alloys used in biomedical applications (Zorn et al., 2011Zorn, G., Baio, J.E., Weidner, T., Migonney, V., Castner, D.G. (2011). Characterization of Poly(sodium styrene sulfonate). Thin Films Grafted from Functionalized Titanium Surfaces. Langmuir 27, 13104-13112. https://doi.org/10.1021/la201918y.; Bauer et al., 2013Bauer, S., Schmuki, P., von der Mark, K., Park, J. (2013). Engineering biocompatible implant surfaces: Part I: Materials and surfaces. Prog. Mater. Sci. 58 (3), 261-326. https://doi.org/10.1016/j.pmatsci.2012.09.001.). As regards the TiAlV alloy, the synthesis of nanostructured layers of TiO₂, obtained by anodization has been proposed to achieve a better osseointegration, and enhancement of strong, long-lasting bonding between the implant surface and the surrounding bone (Hernández-López et al., 2016Hernández-López, J.M., Conde, A., de Damborenea, J.J., Arenas, M.A. (2016). Electrochemical response of TiO₂ anodic layers fabricated on Ti6Al4V alloy with nanoporous, dual and nanotubular morphology. Corros. Sci. 112, 194-203. https:////doi.org/10.1016/j.corsci.2016.07.021.; Germán-Salló and Strnad, 2018Germán-Salló, Z., Strnad, G. (2018). Signal processing methods in fault detection in manufacturing systems. Procedia Manuf. 22, 613-620. https://doi.org/10.1016/j.promfg.2018.03.089.). A hydrothermal method for producing nanostructures on the TiAlV surface has also been proposed for improving the cellular response (Srivas et al., 2019Srivas, P.K., Kapat, K., Das, B., Pal, P., Ray, P.G., Dhara, S. (2019). Hierarchical surface morphology on Ti6Al4V via patterning and hydrothermal treatment towards improving cellular response. Appl. Surf. Sci. 478, 806-817. https://doi.org/10.1016/j.apsusc.2019.02.039.). Other surface modifications have been made focused on the technical aspects, such as the grafting of bioactive molecules, with enhanced osseointegration properties (Wu et al., 2014Wu, S., Liu, X., Yeung, K.W.K., Liu, C., Yang, X. (2014). Biomimetic porous scaffolds for bone tissue engineering. Mater. Sci. Eng. R Reports 80, 1-36. https://doi.org/10.1016/j.mser.2014.04.001.; Yang et al., 2017Yang, D.H., Moon, S.W., Lee, D.-W. (2017). Surface Modification of Titanium with BMP-2/GDF-5 by a Heparin Linker and Its Efficacy as a Dental Implant. Int. J. Mol. Sci. 18 (1), 229. https://doi.org/10.3390/ijms18010229.) or treatments that permit the incorporation of salts or metals to improve osteogenesis and calcification after implantation (Santander et al., 2014Santander, S., Alcaine, C., Lyahyi, J., Pérez, M.A., Rodellar, C., Doblaré, M., Ochoa, I. (2014). In vitro osteoinduction of human mesenchymal stem cells in biomimetic surface modified titanium alloy implants. Dent. Mater. J. 33 (3), 305-312. https://doi.org/10.4012/dmj.2012-015-r.; Bral and Mommaerts, 2016Bral, A., Mommaerts, M.Y. (2016). In vivo biofunctionalization of titanium patient-specific implants with nano hydroxyapatite and other nano calcium phosphate coatings: A systematic review. J. Cranio-Maxillofacial Surg. 44 (4), 400-412. https://doi.org/10.1016/j.jcms.2015.12.004.).

In the same way, another important development has been driven towards surface modifications to promote a bactericide effect to prevent the adhesion and proliferation of microorganisms, thus reducing the probability of both short and long term infection. One proposal has been to obtain surfaces covered with lysozyme. The use of lysozyme prevented Staphylococcus aureus adhesion, while maintaining high cytocompatibility with the important cell lines (Díaz-Gómez et al., 2018Díaz-Gómez, L., Concheiro, A., Álvarez-Lorenzo, C. (2018). Functionalization of titanium implants with phase-transited lysozyme for gentle immobilization of antimicrobial lysozyme. Appl. Surf. Sci. 452, 32-42. https://doi.org/10.1016/j.apsusc.2018.05.024.). Also, a proposal has been made studying the combined effect of alumina and zinc on aluminum alloys, where their osteogenic and antibacterial aspects have been studied. As a result, it has been found that the Zn-doped Al₂O₃ coating delivers outstanding osteogenesis and antibacterial properties, which makes it a promising candidate for use in bone implants (Weng et al., 2018Weng, Y., Liu, H., Ji, S., Huang, Q., Wu, H., Li, Z., Wu, Z., Wang, H., Tong, L., Fu, R.K.Y., Chu, P.K., Pan, F. (2018). A promising orthopedic implant material with enhanced osteogenic and antibacterial activity: Al₂O₃-coated aluminum alloy. Appl. Surf. Sci. 457, 1025-1034. https://doi.org/10.1016/j.apsusc.2018.06.233.). Following up this line of research, the TiAlV has been coated with MgCu by arc ion plating in order to improve its bioactivity and in particular its antibacterial properties. The MgCu coating degraded and gradually disappeared from the surface up to 14 days´ immersion. The degradation of MgCu coating could effectively kill Staphylococcus aureus with an antibacterial rate of 99% (Yu et al., 2018Yu, X., Ibrahim, M., Lu, S., Yang, H., Tan, L., Yang, K. (2018). MgCu coating on Ti6Al4V alloy for orthopedic application. Mater. Lett. 233, 35-38. https://doi.org/10.1016/j.matlet.2018.08.063.).

Adding reactive elements to the surfaces of titanium alloys, such as Cu, Ce or elements like Si and F may increase the proliferation of osteoblasts while showing a decrease in bacteria (Santos-Coquillat et al., 2018Santos-Coquillat, A., Gonzalez Tenorio, R., Mohedano, M., Martinez-Campos, E., Arrabal, R., Matykina, E. (2018). Tailoring of antibacterial and osteogenic properties of Ti6Al4V by plasma electrolytic oxidation. Appl. Surf. Sci. 454, 157-172. https://doi.org/10.1016/j.apsusc.2018.04.267.). Multifunctional coatings have been proposed to combine the wear-corrosion resistance, and to achieve proper amount osteoblast proliferation and bone matrix secretion while achieving a decrease in bacterial colonization.

However, the challenge of obtaining surfaces, which combine both good compatibility and an adequate degree of bactericide, has still not been achieved satisfactorily in the scientific literature.

In previous research works, the TiO₂/CeO₂ coating on TiAlV has been evaluated as an alternative coating system for lengthening the useful life of TiAlV implants (Tavarez-Martínez et al., 2019Tavarez-Martínez, G.M., Onofre-Bustamante, E., De La Cruz-Terrazas, E.C., Escudero-Rincón, M.L., Domínguez-Crespo, M.A. (2019). Evaluation of TiO2/CeO2 coating on Ti6Al4V alloy in PBS physiological medium using conventional and near field electrochemical techniques. Appl. Surf. Sci. 494, 1109-1118. https://doi.org/10.1016/j.apsusc.2019.07.066.). Based on these good results and with the aim of furthering this research, in this work, heat treatment and posterior chemical conversion have been carried out on TiAlV surfaces to study if biocompatibility of the TiAlV can be improved and bacterial growth inhibited or reduced.

In our research, the cell line chosen has been mouse osteoblasts, responsible for the bone growth as they form a deposit of calcium and phosphorous (Patton and Thibodeau, 2007Patton, K.T., Thibodeau, G.A. (2007). Anatomía y Fisiología. 6th ed., Elsevier, Barcelona.). The bacteria strain chosen was the Staphylococcus epidermis (Gram-positive) opportunistic pathogen cultured in aerobic conditions, due to several features: pathogenicity property, presence on the skin and mucus membranes, and ability to survive in high salt concentration media which contain 7-8% NaCl (Madigan et al., 2003Madigan, M.T., Martinko, J.M., Parker, J. (2003). Brock. Biología de los microorganismos. 10th ed., Pearson Educación, Madrid.). Staphylococcus epidermis forms biofilms that grow on the devices, which are introduced into the body like intravenous catheters and medical prostheses. The biofilm is the main virulence factor of this bacterium, acting as a barrier to antibiotics and host’s immune response.

2. MATERIALS AND METHODS

3. RESULTS

Figure 1 shows the surface topography by AFM of TiAlV in as-reception (A), after thermal treatment (B) and subsequent treatment with Cerium chloride (C). In as-received condition, the lines proper to the surface preparation (grounding) can be seen (Fig. 1A). In the thermally treated sample (TiT) appear crystals of TiO₂ with rutile structure verified by DRX (Fig. 1B) (Tavarez-Martínez et al., 2019Tavarez-Martínez, G.M., Onofre-Bustamante, E., De La Cruz-Terrazas, E.C., Escudero-Rincón, M.L., Domínguez-Crespo, M.A. (2019). Evaluation of TiO2/CeO2 coating on Ti6Al4V alloy in PBS physiological medium using conventional and near field electrochemical techniques. Appl. Surf. Sci. 494, 1109-1118. https://doi.org/10.1016/j.apsusc.2019.07.066.). The surfaces of the samples treated thermically and chemically (TiTCe) are more homogenous due to the cerium oxide deposited in pores and defects left by the thermal treatment. The thickness of the layer of cerium oxide is very thin as could be seen only by XPS (Tavarez-Martínez et al., 2019Tavarez-Martínez, G.M., Onofre-Bustamante, E., De La Cruz-Terrazas, E.C., Escudero-Rincón, M.L., Domínguez-Crespo, M.A. (2019). Evaluation of TiO2/CeO2 coating on Ti6Al4V alloy in PBS physiological medium using conventional and near field electrochemical techniques. Appl. Surf. Sci. 494, 1109-1118. https://doi.org/10.1016/j.apsusc.2019.07.066.).

Table 1 reveals the RMS roughness parameter obtained from the different TiAlV samples by AFM. The roughness increases as a consequence of thermal treatment. The chemical treatment with cerium salts does not introduce any important changes in the roughness of the thermically treated samples.

Osteoblasts MC3T3-E1 cells were added on the different surfaces in order to compare the cell proliferation. Figure 2 shows the osteoblasts MC3T3-E1 cultivated after 1, 3, and 7 days in as-received Ti6Al4V (images A, B and C), thermically treated Ti6Al4V, TiT, (images D, E and F), and thermically and chemically treated TiAlV, TiTCe, (images G, H and I), respectively.

The elongated forms of the osteoblast cell with the extension of the filopodia to approach the other cells, are clearly shown. Good adhesion to the different surfaces is also observed. Figure 3 shows comparatively the two modified and as-received Ti6Al4V surfaces after seven days of culture. In general, the cell proliferation on samples TiTCe is adequate but the confluence is not achieved as occurs in both thermally treated and as-received Ti6Al4V surfaces.

The TiT showed the greatest amount of cell proliferation over testing time. The osteoblasts continued growing considerably on the TiT samples over testing time from the first day of culture, while the TiTCe surfaces showed the least cell proliferation. These data agree with that observed by SEM (Figs. 2 and 3). Although the cell proliferation is less in the TiTCe samples, the osteoblast cell morphology and adhesion to the Ce oxide layer (Fig. 2 photo I) revealed a good health and biocompatibility of the cells and so the absence of any cytotoxic effects. In summary, a good adhesion with an elongated morphology of the osteoblasts was observed on all the surfaces. Results are similar to that obtained by Cerroni et al. (2002)Cerroni, L., Filocamo, R., Fabbri, M., Piconi, C., Caropreso, S., Condò, S.G. (2002). Growth of osteoblast-like cells on porous hydroxyapatite ceramics: an in vitro study. Biomol. Eng. 19 (2-6), 119-124. https://doi.org/10.1016/S1389-0344(02)00027-8. with their experiments on porous hydroxyapatite.

Figure 4 shows the cell proliferation of MC3T3-E1 osteoblasts on TiAlV, TiT and TiTCe. It can be seen that the highest proliferation is obtained for TiT surfaces. However, those TiTCe surfaces treated with Ce decrease considerably the proliferation.

A semi quantitative chemical analysis by EDS on the modified surfaces covered with cells for 1, 3 and 7 days was performed. Figure 5 shows the three regions analyzed: sample surface (yellow), cells (dark blue), and the area between cells filled with extracellular matrix, EM (green). Tables 2, 3 and 4 summarize the chemical analyses on these areas in the as-received Ti6Al4V, thermally treated TiT, and chemically treated TiTCe, respectively after 7 days in culture.

In as-received TiAlV, the surface is composed of Ti, Al and V in accordance with those obtained by optical emission spectroscopy, in wt.%: 6.4 Al, 4.5 V, 0.2 Fe and 88.9 Ti, corresponding to the ASTM F136 standard specification (Table 2). The C content is assigned to the handing of the samples. The chemical analysis on the cells (dark blue) reveals an increase in carbon, oxygen and phosphorous content with respect to the area without any cells (Table 2) and the presence of sulfur on the modified surfaces. These are the main constituents of the phospholipids, which form a high percentage of the biological membranes. In the area between the cells (green), the atomic percentages values of carbon, oxygen and phosphorus are between those of the metallic surface (yellow) and the cells (dark blue).

In the TiT samples, the atomic percentage of oxygen on the surface has increased substantially, (Table 3) due to the thermal treatment.

Finally, the chemically treated samples, TiTCe, appear covered with a thin layer of cerium (Table 4). These results are comparable to those obtained by Brunelli et al. (2005)Brunelli, K., Dabalà, M., Calliari, I., Magrini, M. (2005). Effect of HCl pre-treatment on corrosion resistance of cerium-based conversion coatings on magnesium and magnesium alloys. Corros. Sci. 47 (4), 989-1000. https://doi.org/10.1016/j.corsci.2004.06.016., and they have also corroborated in other research works by XPS (Tavarez-Martínez et al., 2019Tavarez-Martínez, G.M., Onofre-Bustamante, E., De La Cruz-Terrazas, E.C., Escudero-Rincón, M.L., Domínguez-Crespo, M.A. (2019). Evaluation of TiO2/CeO2 coating on Ti6Al4V alloy in PBS physiological medium using conventional and near field electrochemical techniques. Appl. Surf. Sci. 494, 1109-1118. https://doi.org/10.1016/j.apsusc.2019.07.066.). Besides the small percentage of Ce, the atomic percentage of the different elements is practically the same as those of the TiT samples. There is not any difference in the composition of the different areas analyzed with respect to the time. (See the supplementary material, showing the analyses of the different samples under study with MC3T3-E1 osteoblast for 1, 3 and 7 days).

The biocompatibility is generally improved with the new surface modifications of Ti6Al4V alloy, but also it is important to find out whether these modified surfaces show evidence of the inhibition of bacterial reproduction.

The viability of the biofilm of Staphylococcus epidermis ATCC 35983 on the TiAlV, TiT and TiTCe surfaces after 24 hours of growth is shown in Fig. 6. It can be observed that the thermal treatment decreases the bacterial viability and that this decrease is greater when they are coated with cerium oxide.

The Fig. 7 shows by scanning electron microscope the appearance of the biofilm architecture formed by the Staphylococcus epidermis ATCC 35983 strain after 24 hours of incubation on the surfaces.

It can be seen that there is a decrease in the bacterial growth on the treated surfaces with respect to the as-received TiAlV. SEM images only provide information about the bacteria adhered to the surface but cannot differentiate between metabolically active and dead bacteria. Nevertheless, a greater number of bacteria not so rounded, apparently damaged on the TiTCe surfaces in comparison to the as-received Ti6Al4V surface can be seen. Some of these bacteria are marked with arrowheads as an example.

4. DISCUSSION

The process of the integration of the implant with the biological tissue, according to Kasemo and Lausmaa (1998)Kasemo, B., Lausmaa, J. (1988). Biomaterial and implant surfaces: a surface science approach. Int. J. Oral Maxillofac. Implants 3 (4), 247-259. and Fathi et al. (2003)Fathi, M.H., Salehi, M., Saatchi, A., Mortazavi, V., Moosavi, S.B. (2003). In vitro corrosion behavior of bioceramic, metallic, and bioceramic-metallic coated stainless steel dental implants. Dent. Mater. 19 (3), 188-198. https://doi.org/10.1016/S0109-5641(02)00029-5., begins with the adsorption of the first water molecules on the surface of the implanted material. These molecules may interact in different ways. If the surface is hydrophilic, the water molecules adhere very strongly but if the surface is hydrophobic so water adheres weakly to the surface. Once the water molecules have interacted with the surface, the ions present in the biological medium, such as Na+ and Cl- among others, are incorporated forming an electrochemical double layer.

In the case of the modified titanium surfaces tested in this work, the sodium and chloride ions were only seen on the TiT and TiTCe samples (Tables 2, 3, 4). According to Patton and Thibodeau (2007)Patton, K.T., Thibodeau, G.A. (2007). Anatomía y Fisiología. 6th ed., Elsevier, Barcelona., these ions dissolved in water contribute to the fluidity of the extracellular matrix.

Subsequently, the proteins and other molecules approach the surface where they are adsorbed creating a layer of a mixture of different proteins in distinct states of conformation. Then, the cells which create interactions with the protein layer by means of the cellular extensions approach the cell membrane and the cell receptors. So, the response of the implanted material-cells depends largely on the type of proteins and their conformation. The result of this interaction may be the integration of the implant or rather the encapsulation of the implant in a fibrous layer or even its rejection.

In our experiments, the atomic percentage of carbon, oxygen and sulfur increased as a consequence of cells growth on materials studied. The sulfur especially increased in the presence of the CeO₂ on the thermally treated surface (Table 4). Shivakumar et al. analyzed the effect of cerium atoms on the synthesis of collagen in cultured cardiac fibroblasts and explants. It was shown that low concentrations of cerium (100 nM) enhanced the synthesis of collagen and high concentrations (100 µM) inhibited it (Fathi et al., 2003Fathi, M.H., Salehi, M., Saatchi, A., Mortazavi, V., Moosavi, S.B. (2003). In vitro corrosion behavior of bioceramic, metallic, and bioceramic-metallic coated stainless steel dental implants. Dent. Mater. 19 (3), 188-198. https://doi.org/10.1016/S0109-5641(02)00029-5.).

In the spaces between the cells, i.e. the extracellular matrix, there is also an increase in carbon and oxygen in comparison with what occurs in the metallic matrix (Tables 2, 3, 4). Proteins are another important component which form the extracellular matrix and whose principal composition is carbon, oxygen and hydrogen (Patton and Thibodeau, 2007Patton, K.T., Thibodeau, G.A. (2007). Anatomía y Fisiología. 6th ed., Elsevier, Barcelona.). The proteins may be structural fibers, glycoproteins or proteoglycans. The structural fibers like collagen and elastin give flexibility to the tissues (Bueno-Vera et al., 2015Bueno-Vera, J.A., Torres-Zapata, I., Sundaram, P.A., Diffoot-Carlo, N., Vega-Olivencia, C.A. (2015). Electrochemical characterization of MC3T3-E1 cells cultured on γTiAl and Ti-6Al-4V alloys. Bioelectrochemistry 106 (Par B), 316-327. https://doi.org/10.1016/j.bioelechem.2015.06.012.). The glycoproteins are protein molecules attached to carbohydrates, among them, the integrins facilitate the adhesion between the cells and the extracellular matrix. The proteoglycans, like chondroitin sulphate, are hybrid molecules, which are made up principally of carbohydrates attached to the skeletons of proteins. In addition, some molecules contain sulfur, iron and phosphorous (Tables 2, 3, 4).

There are several factors that can affect the implant-cell interaction and may inhibit cell growth such as: the ion release and the chemical composition and topography of the surface under study. TiAlV is a material that is capable of passivating instantly with the oxygen in the air, forming a compact, uniform and adherent nano layer of TiO₂, which isolates metallic substrate and reducing considerably the ion release kinetics in very different corrosive media. The chemical composition of the as-received TiAlV alloy favors the formation of a passive layer of TiO₂ of nanometer order. The heat treated alloy has a thicker layer (1 µm) of TiO₂ of rutile while the TiTCe sample has also a thin layer of cerium oxide, in which the Ce is found in two possible Ce³⁺/Ce⁴⁺ oxidation states (Tavarez-Martínez et al., 2019Tavarez-Martínez, G.M., Onofre-Bustamante, E., De La Cruz-Terrazas, E.C., Escudero-Rincón, M.L., Domínguez-Crespo, M.A. (2019). Evaluation of TiO2/CeO2 coating on Ti6Al4V alloy in PBS physiological medium using conventional and near field electrochemical techniques. Appl. Surf. Sci. 494, 1109-1118. https://doi.org/10.1016/j.apsusc.2019.07.066.). The surface chemical composition of the modified Ti substrates is important for the adsorption of proteins. In the case of the TiT samples, TiO₂ is highly hydrophilic, thus favoring the first step towards the metallic material/cell integration (Shivakumar et al., 1992Shivakumar, K., Nair, R.R., Valiathan, M.S. (1992). Paradoxical effect of cerium on collagen synthesis in cardiac fibroblasts. J. Mol. Cell. Cardiol. 24 (7), 775-780. https://doi.org/10.1016/0022-2828(92)93391-V.). In aqueous media, the OH- favors the bond with phosphate ions by means of the Ca²⁺ ions (acting as a bridge) present in the physiological medium. The presence of ions like phosphate or calcium in the culture medium results in a positive stimulus for cell viability and therefore for the process of osseointegration of the implant in the organism. These ions appear in the different table (Tables 2, 3, 4) and some are deposits of calcium and phosphorous which can give rise to the formation of hydroxyapatite, providing the bone with mineralization and strength (Bueno-Vera et al., 2015Bueno-Vera, J.A., Torres-Zapata, I., Sundaram, P.A., Diffoot-Carlo, N., Vega-Olivencia, C.A. (2015). Electrochemical characterization of MC3T3-E1 cells cultured on γTiAl and Ti-6Al-4V alloys. Bioelectrochemistry 106 (Par B), 316-327. https://doi.org/10.1016/j.bioelechem.2015.06.012.). These points serve to anchor the osteoblasts (Burgos Asperilla, 2013Burgos Asperilla, L. (2013). Caracterización y estudio electroquímico de superficies de Ti inmersas en medio fisiológico y en cultivo celular. Tesis PhD, Universidad Autónoma de Madrid.) (Fig. 2).

Most of the heat treatments applied to the TiAlV alloy are carried out in order to obtain a good adherent rutile-based surface with the aim of improving wear, corrosion resistance and biocompatibility. The rutile phase obtained is thermodynamically stable and according to several authors is more inert to a bacterial attack and has greater friction resistance compared to anatase and brockite (Izman et al., 2012Izman, S., Abdul-Kadir, M.R., Anwar, M., Nazim, E.M., Rosliza, R., Shah, A., Hassan, M.A., (2012). Surface Modification Techniques for Biomedical Grade of Titanium Alloys: Oxidation, Carburization and Ion Implantation Processes. In Titanium Alloys. Chapter 9, IntechOpen, Rijeka. https://doi.org/10.5772/36318.). Other studies have shown that the Young modulus of rutile structure is close to that of bone (Ayu et al., 2017Ayu, H.M., Izman, S., Daud, R., Krishnamurithy, G., Shah, A., Tomadi, S.H., Salwani, M.S. (2017). Surface Modification on CoCrMo Alloy to Improve the Adhesion Strength of Hydroxyapatite Coating. Procedia Eng. 184 399-408. https://doi.org/10.1016/j.proeng.2017.04.110.). Despite these encouraging properties, few research works have been reported on the adhesion strength of the oxide layer formed on the titanium substrate.

The smallest cell growth was obtained on the surface of TiTCe samples that is affected by the low affinity of cerium for water (Azimi et al., 2013Azimi, G., Dhiman, R., Kwon, H.-M., Paxson, A.T., Varanasi, K.K. (2013). Hydrophobicity of rare-earth oxide ceramics. Nat. Mater. 12, 315-320. https://doi.org/10.1038/nmat3545.; Tavarez Martínez, 2019Tavarez Martínez, G. de M. (2019). Evaluación de recubrimientos TiO2-CeO2 sobre la aleación Ti6Al4V, mediante técnicas electroquímicas convencionales y de campo próximo en presencia de células vivas. Tesis (Maestría en Tecnología Avanzada), Instituto Politécnico Nacional. http://tesis.ipn.mx/handle/123456789/26595.), which is important in the implant-biological tissue interaction process. This hydrophobic character is due to the electronic structure of the oxides of the rare earths where 4f orbitals (not fully occupied), are protected from the interactions with the surrounding environment by the complete octet of electrons in the external layers 5s²p⁶. Therefore, they have less interaction with water molecules in the interface (Azimi et al., 2013Azimi, G., Dhiman, R., Kwon, H.-M., Paxson, A.T., Varanasi, K.K. (2013). Hydrophobicity of rare-earth oxide ceramics. Nat. Mater. 12, 315-320. https://doi.org/10.1038/nmat3545.). However, it has been reported that by adding cerium oxide on boride surfaces (in the same way we obtained cerium oxide on TiO₂), more uniform, smooth and crack free layers can be obtained, which increase the tribological performance as well as corrosion resistance of the TiAlV (Peng et al., 2018Peng, M.J., Duan, Y.H., Ma, L.S., Shu, B.P. (2018). Characteristics of surface layers on Ti6Al4V alloy borided with CeO₂ near the transition temperature. J. Alloys Compd. 769, 1-9. https://doi.org/10.1016/J.JALLCOM.2018.07.365.; Tavarez-Martínez et al., 2019Tavarez-Martínez, G.M., Onofre-Bustamante, E., De La Cruz-Terrazas, E.C., Escudero-Rincón, M.L., Domínguez-Crespo, M.A. (2019). Evaluation of TiO2/CeO2 coating on Ti6Al4V alloy in PBS physiological medium using conventional and near field electrochemical techniques. Appl. Surf. Sci. 494, 1109-1118. https://doi.org/10.1016/j.apsusc.2019.07.066.). The hydrophobic character of the compounds of Ce on the surfaces is not at all unfavorable, since the soluble compounds of Ce⁴⁺ are associated mainly with a high redox potential of tetravalent cerium ions and the ability to oxidize biological molecules that can cause problems of toxicity. In addition, the Ce³⁺ ions released from the layer at high concentrations may cause a Fenton-like reaction in the biological medium. On the other hand, the nano ceria and cerium ions used in regenerative medicine have shown some interesting properties, as for example, stimulating the growth of fibroblasts and thereby helping the regeneration of the surrounding tissues (Shcherbakov et al., 2020Shcherbakov, A.B., Zholobak, N.M., Ivanov, V.K. (2020). Biological, biomedical and pharmaceutical applications of cerium oxide. In Metal Oxides Series Cerium oxide (CeO2): Synthesis, Properties and Applications. Elsevier, Amsterdam, 279-358. https://doi.org/10.1016/B978-0-12-815661-2.00008-6.).

Other important parameter for cell adhesion and proliferation is the increase in surface roughness of the materials (Anselme and Bigerelle, 2005Anselme, K., Bigerelle, M. (2005). Topography effects of pure titanium substrates on human osteoblast long-term adhesion. Acta Biomater. 1 (2), 211-222. https://doi.org/10.1016/j.actbio.2004.11.009.; Ahmed et al., 2012Ahmed, M.H., Byrne, J.A., Keyes, T.E., Ahmed, W., Elhissi, A., Jackson, J., Ahmed, E. (2012). Characteristics and applications of titanium oxide as a biomaterial for medical implants. in The Design and Manufacture of Medical Devices. Woodhead Publising, Oxford Cambridge Philadelphia New Delhi, pp. 1-57. https://doi.org/10.1533/9781908818188.1.). It has been noted that higher values of surface roughness provide better cell adhesion. It could be seen in Table 1 that the heat treatment increases the surface roughness but the subsequent chemical treatment with cerium salts does not introduce any significant changes in roughness. Accordingly, a greater cell proliferation has been obtained in the samples TiT (Figs. 2, 3 and 4).

In the experiments with bacteria on the modified Ti alloys, the smallest bacterial viability was shown by the surface treated with cerium salts, TiTCe, followed by that treated thermally and after by the as-received state (Fig. 6). The smallest proliferation occurring in the presence of the cerium oxide layer, concurred with other studies carried out by other authors (Pelletier et al., 2010Pelletier, D.A., Suresh, A.K., Holton, G.A., McKeown, C.K., Wang, W., Gu, B., Mortensen, N.P., Allison, D.P., Joy, D.C., Allison, M.R., Brown, S.D., Phelps, T.J., Doktycz, M.J. (2010). Effects of Engineered Cerium Oxide Nanoparticles on Bacterial Growth and Viability. Appl. Environ. Microbiol. 76 (24), 7981 - 7989. https://doi.org/10.1128/AEM.00650-10.; Ciobanu and Harja, 2019Ciobanu G., Harja, M. (2019). Cerium-doped hydroxyapatite/collagen coatings on titanium for bone implants. Ceram. Int. 45 (2), 2852-2857. https://doi.org/10.1016/j.ceramint.2018.07.290.; Shcherbakov et al., 2020Shcherbakov, A.B., Zholobak, N.M., Ivanov, V.K. (2020). Biological, biomedical and pharmaceutical applications of cerium oxide. In Metal Oxides Series Cerium oxide (CeO2): Synthesis, Properties and Applications. Elsevier, Amsterdam, 279-358. https://doi.org/10.1016/B978-0-12-815661-2.00008-6.) where it was shown that the presence of nanocrystals of CeO₂ can inhibit the growth of some bacteria. This effect was explained on the basis of different mechanisms, such as membrane disruption, ROS generation and interference with the nutrient transport functions (Alpaslan et al., 2017Alpaslan, E., Geilich, B.M., Yazici, H., Webster, T.J. (2017). pH-Controlled Cerium Oxide Nanoparticle Inhibition of Both Gram-Positive and Gram-Negative Bacteria Growth. Sci. Rep. 7, 45859. https://doi.org/10.1038/srep45859.). The electron micrographs of the biofilm formation on surfaces (Fig. 7) showed a higher number of not turgid bacteria in the TiTCe surface, possibly due to membrane damage.

Cerium oxide nanoparticles possess anti-inflammatory properties. Nanoceria reduces the production of inflammatory mediators by stimulating the macrophages of mice cell line J774A. In a model of human immunity in vitro, Schanen et al. (2013)Schanen, B.C., Das, S., Reilly, C.M., Warren, W.L., Self, W.T., Seal, S, Drake, D.R. (2013). Immunomodulation and T Helper TH1/TH2 Response Polarization by CeO₂ and TiO₂ Nanoparticles. PLOS ONE 8, e62816. https://doi.org/10.1371/journal.pone.0062816. found that the treatment with CeO₂ nanoparticles induced APCs (antigen-presenting cells) to secrete the anti-inflammatory cytokine, IL-10 and a TH2-dominated T cell profile (Shcherbakov et al., 2020Shcherbakov, A.B., Zholobak, N.M., Ivanov, V.K. (2020). Biological, biomedical and pharmaceutical applications of cerium oxide. In Metal Oxides Series Cerium oxide (CeO2): Synthesis, Properties and Applications. Elsevier, Amsterdam, 279-358. https://doi.org/10.1016/B978-0-12-815661-2.00008-6.).

Nevertheless, according to our research, it is too early to state that cerium oxide has a bactericidal effect. Some factors have to be taken into account such as, the morphology of the nanoceria, the pH of the medium, the nature of the bacteria itself (differences on the surface of the membrane, metabolic differences and/or potential effects on the formation of spores) and, especially, the Ce³⁺/Ce⁴⁺ ratio. An elevated quantity of Ce³⁺ cause an antioxidant effect. Otherwise, if Ce³⁺/Ce⁴⁺ ratio is low, act as an anticarcinogenic and antibacterial agent (Alpaslan et al., 2017Alpaslan, E., Geilich, B.M., Yazici, H., Webster, T.J. (2017). pH-Controlled Cerium Oxide Nanoparticle Inhibition of Both Gram-Positive and Gram-Negative Bacteria Growth. Sci. Rep. 7, 45859. https://doi.org/10.1038/srep45859.).

However, the decrease in bacterial viability, observed in this work, is a good progress, since the increase in hydrophobicity and roughness is normally associated with an increase in bacterial adhesion to surfaces (Donlan, 2002Donlan, R.M. (2002). Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8 (9), 881-890. https://doi.org/10.3201/eid0809.020063.; Boyd et al., 2002Boyd, R.D., Verran, J., Jones, M.V., Bhakoo, M. (2002). Use of the Atomic Force Microscope to determine the effect of substratum surface topography on bacterial adhesion. Langmuir 18 (6), 2343-2346. https://doi.org/10.1021/la011142p.).

5. CONCLUSIONS

ANNEXES

Supplementary material is attached.