287. STRONTIUM AND CYTOKINE MODULATED HUMAN BONE MARROW STROMAL CELLS - A FUTURE TREATMENT FOR OSTEOARTHRITIS?

Abstract

Background: Tissue engineering (TE) strategies using mesenchymal stem cells (MSCs) offer significant potential as a therapeutic approach for treating OA. Local injections of MSCs are currently undergoing Phase IIb/III clinical trials in patients with knee OA. Recently, strontium was shown (SEKOIA study) to slow progression of OA, reduce pain, improve patients' mobility and reduce joint space narrowing. However, the link of increased cardiovascular risk with strontium precludes its use as a first-line disease-modifying osteoarthritis drug (DMOAD) for patients with knee OA. This study examined whether strontium affected osteogenic differentiation of cytokine modulated MSCs and therefore has a potential role as a bioactive molecule for incorporation into future TE strategies for OA. Methods: Human bone marrow stromal cells (HBMSC) were obtained from patients undergoing fractured neck of femur repair (n=3) following ethical approval and informed consent. Cells were cultured in basal media [alpha-modified Eagle's medium (alpha MEM) +1% penicillin/streptomycin+10% fetal calf serum], grown to confluence and seeded at 1×104 cells per well in 24 well plates in either basal or osteogenic (basal+ascorbate+dexamethasone) media, with or without the inflammatory cytokines IL-1 beta (10ng/ml), IL-6 (100ng/ml) and TNF alpha (10ng/ml). Strontium was added to cultures (except controls) at final concentrations of 0.1mM, 1mM or 10mM. Cells were cultured for 7 days, with media changes every 2-3 days. Cell viability was confirmed using cell tracker green and osteogenic differentiation using histological assessment of alkaline phosphatase (ALP) and biochemical assays for ALP and DNA content. Assay results were analysed using one way ANOVA to allow comparison across different culture conditions. Results: Strontium had no adverse effects on the growth of HBMSCs. Optimal cell growth was observed at 0.1-1mM concentrations of strontium. Under control conditions, IL-1 beta produced an additive osteogenic effect, as did IL-6 to a lesser extent. TNF alpha exerted a modest inhibitory effect. This was demonstrated by ALP histochemistry and confirmed through measurement of ALP specific activity. The additive osteogenic effect of IL-1 and IL-6 persisted when strontium was added to the cultures and was most marked at the 1mM concentration. Conclusion: When low concentrations of strontium were added to cytokine modulated HBMSC cultures, the additive osteogenic effects of IL-1 and IL-6 seen in control experiments became more pronounced. Studies in unselected versus Stro-1 selected MSCs will be required to investigate this observation further. Additional in vitro studies in cytokine modulated MSCs will help to clarify whether increased osteogenic differentiation of MSCs in the presence of inflammation contributes to strontium's beneficial effects in OA. The current studies indicate, future TE strategies for OA could then incorporate strontium as a bioactive factor either within a scaffold or delivered in the cell suspension.