Microbial and cell growth on nanoporous anodic titanium oxide (ATO) layers

Magdalena M. Jarosz 1Katarzyna Malec 2Tomasz Sawoszczuk 3Justyna Syguła-Cholewińska 3Iwona I. Wybrańska 2Grzegorz D. Sulka 1Marian Jaskuła 1

1. Jagiellonian University, Faculty of Chemistry, Ingardena 3, Kraków 30-060, Poland
2. Jagiellonian University Collegium Medicum Department of Clinical Biochemistry, Kopernika 15 a, Kraków 31-501, Poland
3. Cracow University of Economics (CUOE), Rakowicka 27, Kraków 31-510, Poland


The most commonly used materials for bone-implants are titanium and its alloys. It is due to their good biocompatibility, high strength to weight ratio and excellent corrosion resistance [1]. Such materials are therefore widely used in orthopedic, dental and other implants, as well as in medical devices (e.g. screws and plates). Unfortunately, there are some drawbacks connected with titanium implants, e.g. their inertness and long-term osseointegration via the natural oxide (TiO2) existing on surface. Therefore, nanoporous materials on Ti surfaces become a novel solution for bone implants [2].

Nanoporous anodic titanium oxide (ATO) layers on Ti formed by electrochemical anodization have been proposed as a potential nanostructured material for bone implants [3-4]. The main advantage of such materials is direct growth of TiO2 on Ti surface, using simple and cost-effective method such as anodic oxidation. Anodization allows to precisely control nanopore size and structure porosity [5]. The presence of nanoporous layer guarantees an excellent inertness of its surface, allowing it to readily heel into the bone tissue.

There are two main aspects connected with bone-implants, osseointegration and risk of post-operative infections. The osseointegration, especially its speed, is a major factor for implant success. The surface topography and surface chemistry are crucial for the short- and long-term success of the osseointegration process. In terms of biomaterial development and implant technology, the cellular response can be affected by topographical structure of the surface. It has been proven that cells sense and react to nanotopography in vitro by exhibiting changes in cell morphology, orientation, cytoskeletal organization, proliferation, signaling and gene expression [6].

One of the most serious side effects connected with implant surgeries is a very high risk of post-operative infection. It is well-known that formation of biofilms by human pathogenic bacteria on medical titanium-based implants can be dramatic, leading to failure of the devices and resulting in the necessity of implant removal [7]. Strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa are reported to be significant contributors to infections associated with orthopedic implants. Hence, finding materials that are less adherent to bacteria, yet good for cell growth are of great importance.

ATO films on Ti foil were prepared via a three-step anodization in glycol ethylene and glycerine based solutions containing NH4F (0.38 wt.%) and H2O (1.79 wt.%) under a constant voltage of 40 V. The duration of first and second anodizing step was 3h. The third step lasted 10 min for glycol ethylene and 1 h for glicerine based electrolytes. The titanium foil (99.5 % purity) was used as both working and counter electrodes. The anodization was performed in two-electrode cells at constant temperature of 20 °C. Some of the samples were then annealed in the furnace in two different temperatures (400 and 1000 °C) for achieving different polymorphic structures of TiO2 (anatase and rutile). One part of the samples were then used for the cell growth, whereas others for microbial examinations. Adipocyte derived stem cells obtained from abdominal liposuction were seeded onto TiO2 surfaces. Cell viability, proliferation and phenotype were assessed by the measurement of redox reactions in the cells, cellular DNA, tritiated thymidine ([3H]-TdR) incorporation and alkaline phosphatase (ALP) production. For the ATP evaluation, the ATPLiteTM kit was used. For the cytotoxicity determination, the CytoTox96® Radioactive Cytotoxicity Assay was used. Staphylococcus aureus bacteria were used for examining an antimicrobial character of nanoporous TiO2 surfaces on Ti. The evaluation of the inhibition of microorganisms growth and possibility of a biofilm formation on various nanoporous TiO2 surfaces were examined. Nanoporous TiO2 surfaces were placed either on the surface of Mueller-Hinton agar which has been inoculated with microorganism before or stroked perpendicularly into the inoculated selected broth. In both techniques the area of growth inhibition on broths was measured. Prior to each experiment, the plates with TiO2 surfaces were sterilized in 170 °C for 2 hours in a Sanyo Sterilizer. The cultivation of microorganisms on broths was carried out in incubators Mermmet at 37 ºC for 24 h. For biofilm formation tests, nanoporous TiO2 surfaces were placed in liquid broths with suspension of microorganism. The cultivation at 37 ºC were continued for 5 days. After that time the occurrence of biofilm on plates was investigated with a microscopic and UV radiation methods.


[1] K.M. Buettner, A.M. Valentine, Chem. Rev., 112 (2012) 1863-1881

[2] M. Stigter, J. Bezemer, K. de Groot, P. Layrolle, J. Control. Release, 99 (2004) 127-137

[3] M. Pisarek, M. Lewandowska, A. Roguska, J. Kurzydlowski, M. Czachor, Mater. Chem. Phys., 104 (2007) 93-97

[4] S-I. Tanaka, H. Tobimatsu, Y. Maruyama, T. Tanaki, G. Jerkiewicz, ACS Appl. Mater. Interfaces, 1 (2009) 2312–2319

[5] G.D. Sulka in: Nanostructured Materials in Electrochemistry, (Ed. A. Eftekhari), Wiley-VCH 2008, pp. 1-116

[6] S. Oh, K.S. Brammer, K.S. Moon, J.M. Bae, S.Jin, Mat. Sci. Eng. C, 31 (2011) 873-879

[7] K.J. Bozic, S.M. Kurtz, E. Lau, K. Ong, T.P. Vail, D.J. Berry, J. Bone Joint Surg. [Am.], 91A (2009) 128–33


Magdalena Jarosz and Katarzyna Malec acknowledge the financial support from the project Interdisciplinary PhD Studies “Molecular sciences for medicine” (co-financed by the European Social Fund within the Human Capital Operational Programme).

This research was partially supported by the Polish Ministry of Science and High Education (Grant No N N204 213340).


Auxiliary resources (full texts, presentations, posters, etc.)
  1. POSTER: Microbial and cell growth on nanoporous anodic titanium oxide (ATO) layers, PDF document, version 1.5, 1.2MB

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Presentation: Poster at Nano-Biotechnologia PL, by Magdalena M. Jarosz
See On-line Journal of Nano-Biotechnologia PL

Submitted: 2012-06-27 15:55
Revised:   2012-06-27 15:55