In Situ Spectroscopic Screening of Osteosarcoma Living Cells on Stoichiometry-Modulated Silicon Nitride Bioceramic Surfaces

Osteosarcoma cell viability, proliferation, and differentiation into osteoblasts on a silicon nitride bioceramic were examined as a function of chemical modifications of its as-fired surface. Biological and spectroscopic analyses showed that (i) postsintering annealing in N₂ gas significantly improved apatite formation from human osteosarcoma (SaOS-2) cells; (ii) in situ Raman spectroscopic monitoring revealed new metabolic details of the SaOS-2 cells, including fine differences in intracellular RNA and membrane phospholipids; and (iii) the enhanced apatite formation originated from a high density of positively charged surface groups, including both nitrogen vacancies (V_N³⁺) and nitrogen N-N bonds (N₄⁺) formed during annealing in N₂ gas. At homeostatic pH, these positive surface charges promoted binding of proteins onto an otherwise negatively charged surface of deprotonated silanols (SiO^-). A dipole-like electric-charge, which includes V_N³⁺/N₄⁺ and SiO^- defective sites, is proposed as a mechanism to explain the attractive forces between transmembrane proteins and the COO^- and NH²⁺ termini, respectively. This is analogous to the mechanism occurring in mineral hydroxyapatite where protein groups are specifically displaced by the presence of positively charged calcium loci (Ca⁺) and off-stoichiometry phosphorus sites (PO₄^2-).