Analysis of bioactivity and cytotoxicity in Ca-P coatings with Si and Ag inclusion obtained by plasma electrolytic oxidation on Ti6Al4V

oxidation on Ti6Al4V S Leal Marin1, H Carreño Garcia2, P Escobar Rivero2, H Estupiñan Duran1. 1 Tribology and surfaces group, Universidad Nacional de Colombia sede Medellín, Colombia 2 Research Center on Tropical Diseases, Universidad Industrial de Santander, Bucaramanga, Colombia. Statement of Purpose: Coatings over Ti6Al4V are extensively studied to improve the compatibility and mechanical properties of the alloy, with emphasis in their uses as raw material for osteosythesis devices. Plasma electrolytic oxidation (PEO) is one of the techniques employed to obtain this kind of coatings, being a highlight technique for its easy technological access, easy sample preparation and be environmental friendly [1,2]. PEO allows ions and particles incorporation into the coating through an electrolytic solution and depends on solution components the coating exhibit different properties. Calcium (Ca) and phosphorus (P) are typical ions included in the electrolytic solution because, regarding with the PEO properties they are deposited into the coating as calcium phosphates, common element in bone substitution and component of the extracellular matrix (MEC) [3]. The aim of this study was analyze the effects of silicon (Si) and silver (Ag) ions incorporation in coatings of Ca-P obtained by PEO. Si has shown ability to be point of nucleation of hydroxyapatite in mineralization process [1,4]. Otherwise, Ag has exhibit antibacterial properties desirable on bone implants to avoid second surgical process and may improve the coating bioactivity due to its metallic nature increasing the electrical conductivity [5]. Methods: Ti6Al4V disk-shaped samples with Ø14mm and 2 mm thick were employed passing through polished, pickled and anodized in orthophosphoric acid to create a stable initial oxide layer. After PEO was performed using a power supply in constant current mode with 70mA/cm2 for 2 minutes. Coatings with two different compositions were obtained, both enriched in Ca2+, PO42ions employing calcium bis(di-hydrogen phosphate)monohydrate, calcium acetate, and ethylene-diamine-tetra-acetic acid disodium as components of the electrolytic solution. In addition, one with inclusion of Si2+ ions, employing sodium silicate and potassium hydroxide in the electrolytic solution and other one without Si2+ ions with a second PEO process using same current conditions for 1 minute. After the PEO Ag2+ ions were included into the coating via electrophoresis with constant voltage, using silver sulphate as reagent. Coatings were characterize with scanning electron microscopy (SEM) to assess the morphological characteristics and with x-ray dispersive energy (EDS) to obtain the chemical elemental composition. In addition, x-ray diffraction (DRX) was used to identify the coating components and electrochemical impedance spectroscopy (EIS) test was performed to establish an equivalent electric circuit of the coat to identify stability and bioactivity. Moreover, biological behavior was assessed with a cell culture of bone osteosarcoma (HOS) (ATCC CRL-1543) of 30.000 cell/mL in culture wells using RPMI-1640 (Gibco) as culture medium supplemented with 10% bovine serum and 1% antibiotics. In a controlled environment of 37°C, 5% CO2 and 95% humidity. Cells were used to analyze cytotoxicity through MTT assay, adhesion through cell count and MEC formation with alkaline phosphatase (ALP) detection. Using culture times of 3 days, 5 days and 9 days, respectively for each test. Results: Coatings obtained analyzed by SEM images showed an interconnected porous morphology with an average size of 2μm and approximately thickness of 4.5μm without Si inclusion and 3μm with Si. Ag inclusion may be observed as small agglomerations of particles (Fig 1b and 1f indicated by arrows). Figure 1 shows coatings morphology for the different coatings obtained changing the composition of the electrolytic solution. No significant changes were observed in the porosity between them.