Nanoscale Morphology of Apatite Precipitated onto Synthetic Hydroxyapatite from Simulated Body Fluid

Dense, polycrystalline, synthetic hydroxyapatite (HA) was incubated for 36 days in modified simulated body fluid (SBF) with increased HCO3 and reduced Cl ion concentrations (27 and 120 mM, respectively) closer to actual blood plasma than typical SBF. The resulting precipitated apatite layer was characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements and found to be nonstoichiometric, calcium deficient (Ca/P~1.06), noncarbonate containing, and of intermediate hydrophilicity (advancing contact angle, θa=76.5±1.3°). The nanoscale surface topography of the SBF-incubated HA sample was imaged by tapping mode atomic force microscopy (TMAFM), observed to be =100 nm in thickness, and composed of three distinct morphologies. These topographically distinct regions were localized within individual grains and facets of the initial HA surface and included: hemispherical, globular structures (maximum lateral dimension, d=44.7±12.7 nm, peak-to-valley height, h=3.6±2.7 nm); elongated, needlelike structures (minimum lateral dimension, w=31.0±8.5 nm, d=104.4±31.1 nm, h=5.0±3.2 nm), and regions of larger, irregularly shaped structures that were relatively smooth (d=504.9±219.1 nm, h=104.0±51.7 nm). Introduction Synthetic hydroxyapatite (HA, Ca5(PO4)3OH), HA-based biomaterials, and HA coatings are used extensively for hard tissue applications due to their bioactivity [1]. Upon implantation in vivo [2] or incubation in vitro in simulated body fluid (SBF) [3], an apatite layer forms on the surface which is considered essential for the nucleation of biological apatite, the promotion of protein adsorption and cell adhesion, and ultimately, the creation of a strong bond with the surrounding tissue [4]. The objective of this study was to directly visualize and quantify the nanoscale topography of apatite precipitated in vitro from SBF onto dense, polycrystalline, phase pure HA using tapping mode atomic force microscopy (TMAFM) imaging, which enables spatial resolutions of <1 nm. New information is presented on the morphological heterogeneity of the apatite layer, as well as the nature of the transition boundaries between topographically different regions. Such a methodology has great potential to contribute insights into the physiochemical mechanisms and temporal evolution of HA interfacial apatite layers and molecular origins of their bone bonding capability. Materials and Methods HA Sample Preparation, Characterization, and Incubation in SBF. Synthetic, phase pure, dense, polycrystalline HA pellets (~1 cm in diameter) were prepared by an aqueous precipitation reaction between calcium hydroxide and phosphoric acid as described previously [5]. The pellets were found to be >98% of the theoretical density (3.13±0.015 g/cm), highly crystalline, phase pure, and approximately stoichiometric (Ca/P ratio=1.67) as measured by water displacement, wide angle X-ray diffraction, and X-ray fluorescence [6]. SBF (total ionic strength=0.155mM, pH 7.4) was prepared with the following ion concentrations (mM); Na(142), K(5), Mg(1.5), Ca(2.5), Cl(120), HCO3(27), HPO4(2.27), SO4(0.5)[7]. NaCl(6.55g), NaHCO3(2.27g), KCl(0.373g), Na2HPO4·2H2O(0.178g), MgCl2·6H2O(0.305g), 37 wt% HCL (5 ml), CaCl2(0.278g), NaSO4(0.071g), and NH2C(CH2OH)3 (Tris buffer,6.055g) were dissolved in that order into 500 mL Millipore water at 37C under continuous magnetic stirring. Millpore water was added to increase the total volume to 1L, the temperature was returned to 37°C, and the pH was balanced to 7.4 using 1M HCL. The solution was filtered with a 0.22 μm vacuum filter and stored in the refrigerator at 4°C until use. The HA pellets were incubated in 100 ml of SBF at 37C for 36 days with no refreshing of the solution. XPS and Contact Angle Measurements. A Kratos AXIS Ultra Imaging X-ray Photoelectron Spectrometer with AlKα X-ray source was used to analyze the initial HA and the precipitated apatite surface compositions at a take-off angle of 0 (penetration depth < 10 nm). Advancing contact angles (θa) were measured using deionized water to assess the wettability of the initial and SBF-incubated HA surfaces (Video Contact Angle System 2000, AST Inc.). Atomic Force Microscopy (AFM). Contact mode AFM (CMAFM) was used to measure the initial HA grain size and TMAFM was employed to image surface topography at higher resolutions, both in ambient conditions with a Digital Instruments Nanoscope IIIA System Controller and Multimode AFM. Thermomicroscopes Si3N4 V-shaped cantilever (end radius, RTIP <50 nm, spring constant, kc~0.01 N/m) and Olympus AC240TS-2 rectangular Si cantilevers (RTIP <10 nm, kc=2 N/m) were used for CMAFM and TMAFM, respectively. Height images were employed to quantify the dimensions of topographical features. Results XPS and Contact Angle Measurements. An XPS survey spectrum on the SBFincubated sample showed the expected Ca(2s,3s,2p), P(2s,2p), and O(Auger,1s,2s) peaks, confirming that the precipitated surface layer was indeed apatite (Fig. 1). Small peaks of Fe, a contaminant from SBF reagents, as well as N and Na directly from SBF reagents, were noted. Na substitution is a possibility. A high resolution scan of the C1s peak revealed typical hydrocarbon contamination and the absence of a carbonate peak at 289.3 eV. The Ca/P ratio was calculated to be 1.06, i.e. Ca-deficient. θa was found to be 76.5±1.3° for the SBFincubated sample, as compared to 65.2±0.85 for the initial HA surface (p<0.005). Fig. 1. XPS spectrum of HA with precipitated apatite layer