Osteoconductive phosphoserine-modified poly({varepsilon}-lysine) dendrons: synthesis, titanium oxide surface functionalization and response of osteoblast-like cell lines

Abstract

The lack of direct bonding between the surface of an implant and the mineralized bony tissue is among the main causes of aseptic loosening in titanium-based implants. Surface etching and ceramic coatings have led to improved osteointegration, but their clinical performance is still limited either by partial bonding or by coating delamination. In this work, a solid-phase synthesis method has been optimized to produce poly(ε-lysine) dendrons, the outermost branching generation of which is functionalized by phosphoserine (PS), a known catalyst of the biomineralization process. The dendrons were deposited onto etched titanium oxide surfaces as a near-to-monolayer film able to induce the formation of a homogeneous calcium phosphate phase in a simulated body fluid over 3 days. The dendron films also stimulated MG63 and SAOS-2 osteoblast-like cells to proliferate at a rate significantly higher than etched titanium, with SAOS-2 also showing a higher degree of differentiation over 14 days. PS-tethered dendron films were not affected by various sterilization methods and UV treatment appeared to improve the cell substrate potential of these films, thus suggesting their potential as a surface functionalization method for bone implants.

Figure 1.

Physico-chemical characterization of G₃ PL-PS dendrons. (a) HPLC and (b) MS. Fragments shown represent (M – CO₂H)³⁺ at 1521.9 m/z, (G3PL – CHN)²⁺ at 956 m/z, M⁵⁺ at 922 m/z and (G3PL+7PS)⁵⁺ at 622 m/z.

Figure 2.

Scanning electron microscopy of (a) unmodified etched TiO₂ surfaces and (b) G₃ PL-PS-functionalized TiO₂ surfaces.

Figure 3.

Surface analysis of TiO₂ surfaces. SEM of (a and b) unmodified etched TiO₂ surfaces, (c and d) PL-PS-functionalized TiO₂ surfaces after 72 h incubation in SBF. Electron diffraction by X-ray analysis of nucleated sites (e) on unmodified etched surface and (f) PL-PS-functionalized TiO₂ surfaces.

Figure 4.

MG-63 osteoblast-like cell adhesion after 3 h incubation on (a) tissue culture plastic, (b) unmodified etched TiO₂ and (c) PL-PS-functionalized TiO₂. Size bar represents 100 µm.

Figure 5.

Osteoblast-like cell proliferation on unmodified etched TiO₂ and PL-PS-functionalized TiO₂ surfaces. (a) MG-63 proliferation on UV-sterilized samples (TC uncoated, tissue culture plastic; TC coated, tissue culture plastic coated with G₃ PL-PS; Ti uncoated, TiO₂; Ti coated, TiO₂ coated with G₃ PL-PS; Ti coated and washed, TiO₂ coated with G₃ PL-PS and washed); (b) SAOS-2 proliferation on UV and gamma-irradiated samples. Tissue culture plastic plates were used as the control. Data were statistically analysed by ANOVA (Tukey's test) and values were considered statistically significant at p ≤ 0.05. Asterisk indicates significantly different samples.

Figure 6.

SAOS-2 osteoblast-like cell differentiation on unmodified etched TiO₂ and PL-PS-functionalized TiO₂ surfaces. Tissue culture plastic plates were used as the control. Data were statistically analysed by ANOVA (Tukey's test) and values were considered statistically significant at p ≤ 0.05. Asterisk indicates significantly different samples.