Effects of multiple sterilization on surface characteristics and in vitro biologic responses to titanium

Abstract

Purpose: This study evaluates the surface changes and effects on in vitro cell attachment and spreading brought about on prepared commercially pure titanium by multiple exposures to common sterilization methods. Materials and Methods: Discs of commercially pure titanium were prepared to approximate the surface roughness of commercially available bone miniplates. Samples underwent sterilization by exposure to ultraviolet light; ethylene oxide sterilization (1, 5, or 10 cycles); or by steam autoclaving (1, 5, or 10 cycles). Representative surfaces from these sterilization groups were examined using a series of surface analytical techniques including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), auger electron spectroscopy (AES), and contact angle measurements. Cell attachment assays using murine fibroblasts were then performed on titanium surfaces from each sterilization group and on tissue culture plastic controls. Sterilized surfaces contained O, C, and N contaminants, which affected surface energetics. Mean percent cell attachment values for each group were obtained for periods of up to 1 hour. Representative samples from each group were examined using SEM to ascertain cell spreading and morphology for each sterilization group. Results: Ultraviolet (UV) sterilized surfaces showed no changes from the unsterilized state macroscopically or under SEM. UV surfaces showed cell attachment levels similar to control surfaces at all intervals, and a chronologic progression of cell spreading. Ethylene oxide-sterilized surfaces showed occasional bluish discoloration and a microscopic particulate contaminant, resulting in modest decreases in cell attachment levels without strong correlation to numbers of sterilization cycles. Autoclaved surfaces generally showed the greatest discoloration and heaviest particulate contamination. Cell attachment levels were lower, and cell spreading was diminished compared with the ethylene oxide-treated group. Conclusions: Both ethylene oxide and steam autoclave sterilization contaminated and altered the titanium surface, resulting in decreased levels of cell attachment and spreading in vitro. Although corroborative in vivo experiments should be conducted, the results of this study indicate that some multiple sterilization regimens for metallic materials may pose serious biologic concerns.