Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability

Abstract

Composite materials based on a titanium support and a thin, alginate hydrogel could be used in bone tissue engineering as a scaffold material that provides biologically active molecules. The main objective of this contribution is to characterize the activation and the functionalization of titanium surfaces by the covalent immobilization of anchoring layers of self-assembled bisphosphonate neridronate monolayers and polymer films of 3-aminopropyltriethoxysilane and biomimetic poly(dopamine). These were further used to bind a bio-functional alginate coating. The success of the titanium surface activation, anchoring layer formation and alginate immobilization, as well as the stability upon immersion under physiological-like conditions, are demonstrated by different surface sensitive techniques such as spectroscopic ellipsometry, infrared reflection-absorption spectroscopy and X-ray photoelectron spectroscopy. The changes in morphology and the established continuity of the layers are examined by scanning electron microscopy, surface profilometry and atomic force microscopy. The changes in hydrophilicity after each modification step are further examined by contact angle goniometry.

Keywords: XPS; alginate; biomimetic surfaces; bisphosphonates; neridronate; poly(dopamine); spectroscopic ellipsometry; surface characterization; surface modification; titanium.

Figure 1

High resolution Ti 2p (left) and O 1s (right) XPS spectra of pristine titanium surfaces (A) and surfaces treated using alkaline piranha (B), 0.5 M NaOH (C), a HCl/H₂SO₄ mixture (D) and piranha solution (H₂SO₄/H₂O₂) (E). The spectrum of flat titanium surfaces deposited on silicon substrates (F) is given for comparison. The dominant contribution (more than 96.7%) within the Ti 2p_3/2 envelope appears at 458.9 ± 0.1 eV and is identified as TiO_2. The measured O 1s spectra (points) was fitted (solid, colored lines) by resolving the individual surface oxide contributions (black lines) centered at 530.3 ± 0.1, 531.5 ± 0.1 and 533.0 ± 0.3 eV arising from titanium oxides (TiO₂ and Ti₂O₃), Al₂O₃ and SiO₂, respectively. The hydroxy groups on the surface gave rise to the peak at 531.8 ± 0.2 eV. The figure also reports the surface density of hydroxy groups (n_OH).

Figure 2

FTIR spectra of starting neridronate (A), APTES (B) and dopamine (C) organic moieties in their native state (red) and corresponding immobilized films on activated flat titanium surfaces (black). The spectra of the immobilized films were taken in IRRAS mode against backgrounds of bare titanium.

Figure 3

High resolution C 1s XPS spectra of neridronate (A), APTES siloxane (B) and PDA (C) films on the surfaces of activated flat titanium substrates (black). The unfilled circles represent the measured data, while the red lines represent the fitted data. The individual contributions to the fitted data of different functional groups present in the films are represented with black lines.

Figure 4

AFM images of the neat, flat titanium surface (R_RMS = 0.5 ± 0.3 nm) (A), and confluent anchor layers of neridronate (R_RMS = 0.5 ± 0.2 nm) (B), APTES (R_RMS = 1.1 ± 0.2 nm) (C) and PDA (R_RMS = 3.6 ± 1.2 nm) (D). The figure also reports AFM images of ALG layers grafted onto neridronate (R_RMS = 0.7 ± 0.3 nm) (E), APTES (R_RMS = 1.8 ± 0.2 nm) (F) and PDA (R_RMS = 2.9 ± 1.0 nm) (G) anchor layers. The AFM measurements on the ALG surfaces were performed on predominantly flat regions with R_RMS values similar to those of the initial anchor layers.

Figure 5

Differential IRRAS spectra of free alginate adsorbed onto a flat titanium surface (A) and covalently bound alginate molecules to the amines of the neridronate (B), APTES (C) and PDA (D) anchor layers. The IRRAS spectra of covalently bound alginate films was characterized by the presence of the carbonyl band (1730 cm⁻¹), amide I (1650 cm⁻¹), amide II band (1540 cm⁻¹) and ν(C–O) stretching modes of the pyranosyl ring, β-(1-4)-glycosidic bonds and hydroxy groups of the polysaccharide (1200–1000 cm⁻¹). The spectra were referenced to corresponding background spectra of bare titanium and titanium bearing different anchor layers.

Figure 6

High resolution C 1s XPS spectra of alginate coatings on neridronate (A), APTES siloxane (B) and PDA (C) anchoring layers. The unfilled circles represent the measured data, while the red lines represent the fitted data. The individual contributions to the fitted data of different functional groups present in the films are represented with black lines.

Figure 7

Ellipsometric thickness and water contact angle evolution of ALG bound to neridronate, APTES siloxane and PDA during the immersion in PBS (37 °C, pH = 7.4) (mean value ± SD, n = 15).

Figure 8

Evolution of IRRAS spectra of ALG bound to neridronate (A), to APTES siloxane (B) and to PDA (C) upon immersion in PBS at 37 °C for 7 days.

Scheme 1

Performed surface treatments and subsequent reactions for the activation and modification of titanium surfaces. (A) Cleaning and activation procedures for the removal of inorganic and organic contaminants from the titanium surface and to increase the number of free hydroxy groups. (B) Immobilization of neridronate, APTES siloxane and poly(dopamine) anchor layers through surface specific reactions between the phosphonate, silane and catechol groups of corresponding compounds and hydroxy groups on the surface. (C) Covalent binding of ALG chains to amino groups present in the anchor layers by the EDC/NHS coupling reaction.