METHODS: Polished titanium discs (10x10x5mm) were pre-treated with 30% H2O2 (5ml H2O2/g disc) at room temperature for 0, 1, 3, 6 or 24h and 1, 2 or 4wks. Rat bone marrow-derived osteoprogenitor cells were cultured, with supplements for 7 days, to induce differentiation. Osteoblast-like cells were seeded onto H2O2 pre-treated titanium discs (5x103cells/cm2, 3 discs/treatment) and maintained at 37°C/5% CO2, for 5 days. Osteoblast-like cells were assessed for viability (MTT), morphology (Scanning Electron Microscopy), adherence (ethidium bromide/acridine orange, Fluorescent Microscopy) and proliferation ([3H]-thymidine).
RESULTS: All H2O2-treated discs maintained osteoblast-like cell viability, irrespective of H2O2 pre-treatment, with no significant differences between pre-treatments (p>0.05). Cells on untreated (control) and 1-24h H2O2-treated discs exhibit a spherical/flattened morphology. Cells on 1-4wk H2O2-treated titanium discs appeared more irregular/polygonal. No significant differences in cell attachment/proliferation on H2O2-treated discs were observed, up to 24h, compared to controls (p>0.05), although significant decreases in cell attachment/proliferation were evident on 1-4wk-treated discs (p<0.05). CONCLUSIONS: As an optimal surface roughness range to enhance cellular adherence is well-established, it appears that H2O2 pre-treatments of 1-4wks, with extremely rough surfaces, are detrimental to cellular adherence/proliferation. However, such surfaces enhance osteoblast-like cell differentiation, as supported by cell morphology. In order to improve cellular activity for osseointegration, it should be considered whether it preferable to H2O2 pre-treat titanium for under 24h, to induce cell adherence/proliferation and delay differentiation, or over 24h, to induce cell differentiation, whilst reducing adherence/proliferation?