Crystallographically engineered, hydrothermally crystallized hydroxyapatite films: an in vitro study of bioactivity

The aim of this study was to evaluate the bioactivity of hydroxyapatite films composed of hexagonal single crystals that display {1010} and {0001} crystallographic faces. The effect of engineered [0001] crystallographic orientation was investigated in parallel. Films were deposited by triethyl phosphate/ethylenediamine-tetraacetic acid doubly regulated hydrothermal crystallization on Ti6Al4V substrates (10, 14, 24 h). Bioactivity was investigated by analysis of MC3T3-E1 pre-osteoblast spreading using scanning electron microscopy and quantitative analysis of cell metabolic activity (Alamar Blue) (0-28 days). Scanning electron microscopy and X-ray diffraction were used to evaluate the ability of films to support the differentiation of MC3T3-E1 pre-osteoblasts into matrix-secreting, mineralizing osteoblasts. Results demonstrated that all films enabled MC3T3-E1 cells to spread, grow, and differentiate into matrix-secreting osteoblasts, which deposited biomineral that could not be removed after extraction of organic material. Differences in [0001] HA crystallographic orientation were not, however, found to significantly affect bioactivity. Based on these results, it is concluded that these hydrothermal hydroxyapatite films are non-toxic, bioactive, osteoconductive, and biomineral bonding. The lack of a relationship between reported hydroxyapatite crystallographic face specific protein adsorption and bulk HA bioactivity are discussed in terms of crystallographic texture, surface roughness, assay robustness, and competitive protein adsorption.