Application of FIB‐SEM tomography for analyses of ceramic coatings deposited on titanium alloy and material‐cell interface

Abstract

Biomedical engineering is a fast developing field of medicine, employing biomaterials to replace or augment damaged and diseased tissues. Growing population with osteoarthritis problems and extending life span contributes to increased demand for effective solutions in arthroplasty. Titanium alloys are the materials of the choice in case of elements of artificial joints. They are implanted directly into the bones. Long lasting performance of those implants depends on their integration with surrounding tissues. Surface modification of titanium alloys is performed to improve osseointegration. It can be achieved by deposition of porous, ceramic coatings. Such a structure facilitates bone cells ingrowth and stable anchorage of the implant in the bone. Understanding of the reactions at the interface between materials and cells will help to optimize the design of new implants with improved performance. In current work, a titanium alloy (Ti6Al7Nb) was used as a substrate in micro‐arc oxidation (MAO) process. As the effect of this electrochemical reaction the protective and bioactive coating composed of titanium dioxides, calcium titanate and calcium phosphates was formed on the metallic sample. The obtained complex ceramic, porous layer was investigated by SEM and SEM‐EDS (Merlin Gemini II, Zeiss) and FIB‐SEM tomography (NEON CrossBeam 40EsB, Zeiss) to analyze microstructure, chemical composition and internal structure, including pores network. In vitro cell culture is a standard method to evaluate biocompatibility of new or modified materials. Thus, osteoblast‐like cells line MG‐63 were cultured for three days at the surface of coated titanium alloy samples. Subsequently cells attached to the ceramic coating were fixed, dehydrated and gold sputtered for electron microscopy investigations. Cells shape and distribution at rough and porous structure of the coating containing hydroxyapatite (HA) in the outer layer was investigated based on plain view SEM images (Fig. 1a). Area prepared for tomography by FIB milling of surrounding material is presented at Fig. 1b (marked by red square). FIB‐SEM tomography enables to see both material surface and cells at nanometer scale (Fig. 1c). The 3D reconstruction allows for visualization and investigations of the interface between coated material and cell in the analyzed sample volume.