Abstract
Introduction
The osteogenic capability of any biomaterial is governed by a number of critical surface properties such as surface energy, surface potential, and topography. Prior work suggested that the Si-Y-O-N phase(s) present in the form of a thin (<150 nm), interrupted film at the surface of an annealed silicon nitride bioceramic may be responsible for an observed upregulation of osteoblastic activity due to passive surface properties and dissolution of chemical species. In this study high- resolution analytical electron microscopy was utilized to identify the Si-Y-O-N phase present on the annealed silicon nitride surface, and dissolution studies were employed to elucidate mechanisms of the material's favorable cell interactions.
Materials and Methods
Si3N4 discs (12.7 mm diameter × 1 mm thick) containing Y2O3 and Al2O3 sintering aids were processed using conventional techniques and subsequently subjected to annealing in a nitrogen atmosphere. Pre-cultured SaOS-2 osteosarcoma cells at a concentration of 5 × 105 cells/ml were seeded onto sterile polished nitrogen-annealed Si3N4 discs in an osteogenic medium consisting of DMEM supplemented with about 50 µg/mL ascorbic acid, 10 mM β-glycerol phosphate, 100 mM hydrocortisone, and 10% fetal bovine calf serum. The samples were incubated for up to 7 days at 37°C with two medium replenishments. Transmission electron microscopy (TEM) images were acquired from focused ion beam (FIB)-prepared samples using a Hitachi HF-3300 TEM (300 kV). Scanning transmission electron microscopy (STEM) images were recorded using a Nion UltraSTEM 100 (60 kV). STEM high-angle annular dark-field (HAADF) imaging and energy dispersive X-ray spectroscopy (EDS) analyses were performed on a JEOL JEM2200FS (200 kV) equipped with a third-order CEOS aberration corrector and a Bruker XFlash silicon drift detector.
Results
A cross-section of the of the Si3N4/extracellular polymer (ECP) interface is illustrated in Fig. 1(a)∼(b) as a high- angle annular dark field (HAADF) STEM image (a) with and EDS map overlay (b) highlighting locations of Ca, Y, and Si. The underlying Si3N4 microstructure is covered by a yttrium-rich intergranular phase (IGP) film. Deposition of cell-derived hydroxyapatite (HAp) occurred directly onto this IGP film. In Fig. 2, a bright field TEM image (electron diffraction pattern inset) shows the interface between the partially-crystalline HAp and the Y-Si-O-N phase, identified as monoclinic yttrium disilicate (i.e., m-Y2Si2O7) with a 2 atomic% N impurity, at teh atomic scale. Although rapid electron damage of the mineralized ECP was observed, EDS analyses suggested a Ca/P ratio of ∼1.43, along with the incorporation of Si.
Conclusions
The osteogenic Si-Y-O-N phase was successfully identified as a minority concentration of Si3N4 dissolved into a m-Y2Si2O7 matrix. Evidence of the release of (SiO4)4− tetrahedra from this phase into the local biological microenvironment and their incorporation into the cell-derived HAp layer was also observed. Identification of this phase paves the way for ongoing work to understand and optimize this novel biomaterial.
For any figures or tables, please contact the authors directly.
