Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides

Melo Rodriguez, Gabriela; Bowen, James; Grossin, David; Ben-Nissan, Besim and Stamboulis, Artemis (2017). Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides. Colloids and Surfaces B: Biointerfaces, 160 pp. 154–160.



Surface modifications are usually performed on titanium alloys to improve osteo-integration and surface bioactivity. Modifications such as alkaline and acid etching, or coating with bioactive materials such as hydroxyapatite, have previously been demonstrated. The aim of this work is to develop a peptide with combined titanium oxide and hydroxyapatite binders in order to achieve a biomimetic hydroxyapatite coating on titanium surfaces. The technology would also be applicable for the functionalisation of titanium and hydroxyapatite surfaces for selective protein adsorption, conjugation of antimicrobial peptides, and adsorption of specialised drugs for drug delivery. In this work, functionalisation of Ti6Al4V and hydroxyapatite surfaces was achieved using combined titanium-hydroxyapatite (Ti-Hap) peptides based on titanium binder (RKLPDA) and hydroxyapatite binder (EEEEEEEE) peptides. Homogeneous peptide coatings on Ti6Al4V surfaces were obtained after surface chemical treatments with a 30 wt % aqueous solution of H2O2 for 24 and 48 hours. The treated titanium surfaces presented an average roughness of Sa=197 nm (24 h) and Sa=128 nm (48 h); an untreated mirror polished sample exhibited an Sa of 13 nm. The advancing water contact angle of the titanium oxide layer after 1 hour of exposure to 30 wt % aqueous solution of H2O2 was around 65°, decreasing gradually with time until it reached 35° after a 48 hour exposure, suggesting that the surface hydrophilicity increased over etching time. The presence of a lysine (L) amino acid in the sequence of the titanium binder resulted in fluorescence intensity roughly 16 % higher compared with the arginine (R) amino acid analogue and therefore the lysine containing titanium binder was used in this work. The Ti-Hap peptide KKLPDAEEEEEEEE (Ti-Hap1) was not adsorbed by the treated Ti6Al4V surfaces and therefore was modified. The modifications involved the inclusion of a glycine spacer between the binding terminals (Ti-Hap2) and the addition of a second titanium binder (KKLPDA) (Ti-Hap3 and Ti-Hap4). The Ti-Hap peptide aptamer which exhibited the strongest intensity after the titanium dip coating was KKLPDAKKLPDAEEEEEEEE (Ti-Hap4). On the other hand, hydroxyapatite surfaces, exhibiting an average roughness of Sa=1.42 µm, showed a higher fluorescence for all peptides compared with titanium surfaces.

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