Due to its remarkable stoichiometric flexibility and surface chemistry, hydroxyapatite (HAp) is the fundamental structural material in all vertebrates. Natural HAp's properties inspired an investigation into silicon nitride (Si3N4) to see if similar functionality could be engineered into this bioceramic. Biological and in situ spectroscopic analyses were used to monitor the response of osteosarcoma cells (SaOS-2) to surface-modulated Si3N4 and a titanium alloy after long-term in vitro exposure.
Materials and Methods
Four groups of Si3N4 discs, Ø12.7×1.0mm, (Amedica Corporation, Salt Lake City, UT USA) were subjected to surface treatments: (i) “As-fired;” (ii) HF-etched (5% HF solution for 45 s); (iii) Oxidized (1070°C for 7 h); and (iv) Nitrogen-annealed (1400°C for 30 min, 1.1 bar N2 gas).1 Titanium alloy discs (Ti6Al4V, ASTM F136) were used as a control group. SaOS-2 cells cultured for 24 h at 37°C were deposited (5×105 cells/ml) and incubated on the UV sterilized discs in an osteogenic medium for 7 days at 37°C. Cell proliferation was monitored using scanning electron and laser microscopy. The Receptor Activator of NF-kB Ligand (sRANKL) and the insulin growth factor 1 (IGF-1) were used to evaluate osteoclast formation and cell proliferation efficiency, respectively. In situ Raman spectroscopy was employed to monitor metabolic cell activity. Statistics (n≥3) were analyzed using the Student's t-test or one-way Analysis of Variance with p<0.05 considered significant.
Results are presented in Figure 1(a)∼(c) for HAp formation, free sRANKL, and IGF-1, respectively. These data indicate that the N2-annealed Si3N4 samples had the highest amount of HAp formation followed by the as-fired, oxidized, and HF samples. The Ti-alloy showed moderate HAp formation; but it had a higher amount of free sRANKL as compared to all Si3N4 samples. These data suggest that the Si3N4 represented a friendlier environment for SaOS-2 cell differentiation. The IGF-1 concentration did not differ among the Si3N4 samples, but they were all higher than the Ti-alloy. Higher IGF-1 stimulates cells to proliferate and differentiate.2 In Figure 2, in situ collected Raman spectra confirmed enhanced formation of HAp on the Si3N4 samples, especially the N2-annealed material.
Enhanced apatite formation was found to originate from a high density of positively charged surface groups, including nitrogen vacancies (VN3+) and nitrogen N-N bonds (N4+).3 These surface charges promoted binding of proteins onto the negatively charged Si3N4 surface. A dipole-like-charge of VN3+/N4+and SiO− defective sites is proposed as a mechanism to explain the attraction between proteins and the COO− and NH2+ terminus, respectively. This is analogous to the mechanism occurring in hydroxyapatite where protein groups are displaced by positively charged calcium loci (Ca+) and off-stoichiometry phosphorus sites (PO42−).
Osteoblast proliferation and apatite-growth are important properties in regenerative bone therapies. In general, these properties were pronounced on all of the Si3N4 substrates; but achieved maximum values on the N2-annealed Si3N4.