Abstract
Introduction
Acute spinal cord injuries (SCIs) cause significant worldwide morbidity and mortality, with an overall prevalence of ~750 injuries per 1,000,000 people. Currently no pharmacologic adjuvant has been repeatedly demonstrated to lead to improved neurologic outcomes after these injuries, however preliminary studies suggest that Riluzole, a sodium channel-blocking medication, may be neuroprotective in patients with an acute SCI. Because long-term outcomes after an SCI may be compromised if a solid fusion is not achieved, it is critical to determine the effect of riluzole on bone formation. This study investigated the effects of riluzole on human bone marrow derived mesenchymal stromal cells (MSCs) and human primary osteoblasts (OBs).
Material and Methods
For cell viability testing MSCs and OBs were seeded in 96-well plates at 7,500 cells/cm2. After 24 hour, the cells were treated with osteogenic medium containing different concentrations of riluzole (50 ng/mL; 150 ng/mL; 450 ng/mL). Control groups of MSCs and OBs were cultured without riluzole. After two and seven days, cell viability was determined using the Cell Titer Blue assay (Promega).
For quantification of Alkaline Phosphatase (ALP) activity, MSCs and OBs were plated in 24-well plates (10,000 cells/cm2) and cultured for 2 days in basal medium. Then they were treated with standard osteogenic differentiation medium. Control groups and experimental groups that were exposed to different concentrations of riluzole (50 ng/mL; 150 ng/mL; 450 ng/mL) were cultured. After 7, 14, 21 and 28 days, the cell layers were extracted with 0.1%Triton-X in 10 mM Tris-HCl (pH 7.4), and ALP activity was measured using the Sigma Kit (No.104) according to the technical protocol. ALP activity was normalized to the DNA content of the respective sample (CyQuant, Invitrogen). All experiments were performed in triplicate for two MSC donors and two OB donors.
Results
The applied concentrations of riluzole had no influence on the cell viability of MSCs or osteoblasts after seven days. There was also no clear effect of riluzole supplementation on the proliferation of the MSCs or OBs. In MSCs, the ALP activity per DNA appeared to peak one week earlier with lower doses of riluzole compared with the highest dose of riluzole used in the study; while this trend was not observed in the case of OBs. For MSC donor 1, highest levels of ALP/DNA were noticed at day 7 (70.2 ± 30.4 and 60.8 ± 11.9 mmol/g*min for 50 and 150 ng/mL riluzole, respectively); for MSC donor 2, ALP/DNA levels peaked at day 21 (43.0 ± 8.6 and 80.6 ± 26.5 mmol/g*min) for 50 and 150 ng/mL riluzole groups, while in the 450 ng/mL group highest levels were observed at day 28.
Conclusion
Early clinical trials suggest that riluzole may lead to improved neurologic function in patients who sustain an acute SCI, and the results of the current study suggest that low dose riluzole has no effect on the viability or function of either MSCs or OBs; however, the delayed peak of ALP in MSCs exposed to a high dose of riluzole may indicate that high doses of riluzole may slow osteogenic differentiation.