Nitric Oxide Release Enhances Osteogenic Effects of Diethylenetriamine and Spermine

Objectives: Nitric oxide (NO) is a positive modulator of osteogenesis shown to increase bone formation. Local controlled release may result in enhanced bone healing. Objective: Evaluate the effects of NO releasing small molecules diethylenetriamine (DETA) and spermine on osteogenesis in an MC3T3-based in vitro model.
Methods: MC3T3-E1 Subclone-4 cells were cultured in media containing 1 or 50 μM DETA or spermine [NO releasing (+NO) and polymer only controls (-NO)] and tested for cytotoxicity (4 and 24-hours, n=6), proliferation (days 3-9, n=6), calcium accretion (alizarin-red) and calcium quantification (days 21 and 28, n=4), collagen organization by picrosirius red (PSR, days 14 and 21, n=3), and R-T-PCR for osteogenic markers RUNX2, COL1α1, BSP, OSX, and OCN. Statistical significance: One-way ANOVA Bonferonni post-hoc analysis (p<0.05).
Results: DETA and spermine (+ NO) were not toxic at 1 μM for 4 and 24-hour timepoints. Spermine was toxic at 50 μM and this dose was discontinued for spermine. DETA (+ NO) was not toxic at 50 μM. DETA (+NO) led to a small decrease in proliferation at 50 μM, no change in proliferation was detected at 1 μM for DETA and spermine (+ NO). Alizarin staining and calcium quantification demonstrated spermine itself led to increased calcium, with a further increase with +NO release. DETA itself did not increase calcium but did with +NO release. PSR data agreed and showed an increase in red signal (more mature collagen) for spermine, with a further increase with +NO, and an increase in red for DETA (+NO only). RT-PCR data showed increases in COL1α1, OSX, and OCN related to DETA and spermine themselves, and not addition of NO release.
Conclusions: We have demonstrated that DETA and spermine alone affect osteogenesis, with some further positive effect when engineered to release NO. These results are promising for development of treatments for bone restoration using NO releasing small molecules.