Stability of Surface‐Enhanced Ultrahydrophilic Metals as a Basis for Bioactive rhBMP‐2 Surfaces

Abstract

For several years the treatment of metals like cp titanium and 316L stainless steel with concentrated chromosulfuric acid at high temperatures (230)(231)(232)(233)(234)(235)(236)(237)(238)(239)(240) C) has formed the basis for preparing ultra-hydrophilic priming coats on these metals (Jennissen et al. Materialwiss. Werkstofftech. 30, 838-845, 1999). Metals treated in this way have been called surface-enhanced, displaying a characteristic ultrastructure, and can be easily modified to carry a biocoat of recombinant human bone morphogenetic protein 2 (rhBMP-2). The major oxide on surface enhanced titanium is TiO 2 . Thus this TiO 2 -layer could be responsible for the ultra-hydrophilic properties of the priming coat. Irradiation of TiO 2 layers by ultraviolet light (Wang et al., Nature 388, 431-432, 1997) has been shown to endow these layers with ultra-hydrophilic properties (i.e. contact angles of $ 0). However the ultra-hydrophilic TiO 2 -layers produced by irradiation are unstable and revert to the original high contact angles of $ 70 within several days. The question of whether the ultra-hydrophilic surfaces prepared by the chromosulfuric acid method show long-term stability was therefore important to answer. In addition the question if rhBMP-2 immobilized on such a surface will retain its biological activity was of great interest. In this paper it will be shown that ultrahydrophilic titanium mini-plates retain their ultra-hydrophilicity with contact angles of 0-8 unchanged for at least 50 days and support the immobilization of rhBMP-2 in a biologically active form.

Chemical Functionalization of a Hydroxyapatite Based Bone Replacement Material for the Immobilization of Proteins

“…After fixative was removed by the three brief washes with phosphate buffer, cell membranes were permeabilized by washing with DPBS/ Tween 20 0.2 % (v/v) and were then rinsed with phosphate buffer. The endogenous phosphatase detection kit ELF-97 (Substrate: 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; Molecular Probes, Inc., Oregon, USA) was employed in the cell culture experiments according to the manufacturer's instruction (see [17]). The resulting product forms an intensely fluorescent yellow-green precipitate at the site of enzymatic activity.…”

Preparation and properties of ultra‐hydrophilic surfaces on titanium and steel

“…As has been reported previously [24] a nanostructure may form additionally on titanium surfaces as a result of chromosulfuric acid action (see also [22]). These nanostructures can vary depending on the type of titanium employed, its pretreatment and the conditions of chromosulfuric acid etching itself.…”

“…100 nm in diameter and very closely packed. Previous work indicated by the Rockwell test, that these structures are firmly fixed to the metal in the surface [22].…”

“…0.15 M NaCl [20]. We have however shown that ultra-hydrophilic titanium surfaces (also 316L steel and CoCrMo surfaces) can be stored for extended periods of over four years [24] in dry methanol or dry ethanol [22] without losing their ultra-hydrophi- lic properties. These surfaces are also stable for extended times in dry acetone and in aqueous solutions of 1 M NaCl (unbuffered) (not shown).…”

“…Temperature gradient method. Previously the standardized method for surface enhancing of transition metal surfaces by chromosulfuric acid was based on a moderate temperature gradient, which resulted from a "cold-loading/cold unloading" procedure [2,18,22]. In this method the metal miniplates in a small rack made either of Teflon or titanium are immersed in "cold" (i.e.…”