Synthesis and Characterization of Biomimetic Hydroxyapatite Nanoconstruct Using Chemical Gradient across Lipid Bilayer

In this study, we synthesized biomimetic hydroxyapatite nanoconstruct (nanosized hydroxyapatite, NHAp) using a double emulsion technique combined with a chemical gradient across a lipid bilayer for surface modification of a titanium (Ti) implant. The synthesized NHAp was characterized by dynamic light scattering, X-ray diffraction, transmission electron microscopy, and Fourier transform infrared (FTIR) spectroscopy, and it was further tested for its biocompatibility and in vitro proliferation efficacy using normal human osteoblasts (NHOst). The results showed that the synthesized NHAp had a hydrodynamic diameter of ∼200 nm with high aqueous stability. The chemistry of the NHAp was confirmed by FTIR spectroscopic analysis. Typical FTIR vibrational bands corresponding to the phosphate group (PO4(3-)) present in hydroxyapatite (HAp) were observed at 670, 960, and 1000 cm(-1). A broad band at 3500 cm(-1) confirmed the presence of a structural -OH group in the NHAp. Powder X-ray crystallographic diffraction further confirmed the formation of NHAp with characteristic reflections in (002), (211), (130), and (213) planes at respective 2θ degrees. These reflection planes are similar to those of typical HAp crystallized toward (002) and (211) crystallographic planes. The mechanism of the formation of NHAp was studied using the fluorescence resonance energy transfer (FRET) technique. The FRET study showed the fluorescent recovery of a donor fluorophore and the mechanism of the insertion of lipids into nanodroplets obtained from the first water-in-oil (w/o) emulsion during the formation of the second oil-in-water (o/w) emulsion. With these confirmations, we further studied NHOst cell proliferation on a Ti surface. When NHOst were cultured on the Ti surface coated with the NHAp, a distinct proliferation pattern and cell-cell communication via cytoplasmic extension on the substrate surface were observed. In contrast, a bare Ti surface showed diminished cell size with minimal adherence. This result indicates that our NHAp covered with a phospholipid bilayer provides a proper environment essential for cell adhesion, which is especially important for bone implants, and the inclusion of NHAp on the Ti substrate would be an effective support for long-term sustainability of implants.