AFM, SEM and epifluorescence microscopy of the Cu-polymer coating-cell culture interface

GRILLO C.A.; BERTUOLA M.; FERNÁNDEZ LORENZO M.; PRIETO E.

La Plata

Workshop; Imaging techniques for biotechnology and biomedical applications; 2016

CCT-CONICET de la ciudad de La Plata, INIFTA, Ecola Polytechnique Federale de Laussane, MINCyT, AGENCIA

Polymeric coatings (PCs) can be used to reduce the release of corrosion products and enhance biocompatibility of degradable metallic materials (DMM). Different microscopic techniques can be used to evaluate the process occurring at this PC-DMM/biological medium interface. The aim of this work is to investigate the surface characteristics of two PCs formed on copper surfaces by a combination of nanoscopic/microscopic techniques: Atomic Force Microscopy (AFM) and Environmental Scanning Electron Microscopy (ESEM). Polymeric layers of carvacrol (polyCarvCu) and thymol (polyTOHCu), were formed by electropolymerization of these phenolic compounds on copper surfaces by cyclic voltammetry in hydroalcoholic alkaline solution [1?3]. Additionally, the impact of the protective layers on the biological medium was analyzed in MC3T3-E1 cell line culture by viability (Acridine Orange staining) and clonogenic assays using Epifluorescence Microscopy (EM). Tapping® mode AFM in topographic mode was used to characterize the substrates, using silicon tips. Nanoscope 7.30 and Nanoscope Analysis 1.5 software were employed to obtain the images and ESEM images of polyCarvCu and polyTOHCu were taken using an environmental scanning electron microscope FEI Quanta 200. AFM images (Figure 1) evidenced that Cu surface was covered by a closed packed layer and round nanoflakes can be distinguished on this surface. PolyCarvCu roughness was less than that of polyTOHCu (Rmax=54 nm and 161 nm, respectively). The line profile of the AFM image suggests that particles may be less than 5 nm height in case of polyCarvCu while 35 nm globular particles that form a cauliflower like structures [4], were distinguished for polyTOH. SEM images showed a homogeneous layer in case of polyCarv and a rough surface in case of polyTOH.EM analysis is an appropriate tool to follow cell viability. It was found that after 24 h exposure uncoated Cu showed only 28.88% while polyTOHCu reached 72% and polyCarv was close to the viability of the control (100%) (p