Biotinylation of silicon-doped hydroxyapatite: A new approach to protein fixation for bone tissue regeneration

Silicon-doped hydroxyapatite has been functionalized with biotin molecules as a new methodology for the attachment of proteins, peptides or growth factors through the formation of avidin–biotin complex in this material. Bioceramic biotinylation has been performed by esterification reaction between the OH groups of hydroxyapatite and COOH groups of biotin molecules. Several parameters of the biotinylation, such as the addition of N,N′-dicyclohexylcarbodiimide (DCC), the biotin/bioceramic molar ratio and the activation time, have been studied in order to improve both the amount of anchored biotin on the bioceramic surface and its bond strength. The grafting of biotin on a silicon-doped hydroxyapatite surface was determined using 13C nuclear magnetic resonance, Fourier transform infrared spectroscopy and elemental analyses. The results show that the addition of DCC significantly increases both the amount of biotin grafted and the bond strength, because the major part is through covalent bonding. Lixiviation studies in simulated body fluid (SBF) at 37 °C have confirmed such results, showing that the retention grade after 7 days in SBF was of ca. 63%. Fluorescein isothiocyanate–avidin complexation has been performed on three-dimensional (3-D) scaffolds prepared by a rapid-prototyping technique. Confocal microscopy studies show a homogeneous distribution with a higher incorporation rate of the protein over the entire external surface of the biotinylated 3-D scaffold.