Abstract
Objective
Human bone marrow stromal cells (HBMSCs) are multipotent and can form bone, cartilage or other tissues under different inductive conditions. The aim of this study was to investigate the effects of enamel matrix derivative (EMD) on the growth and osteogenic differentiation of HBMSCs.
Methods
HBMSCs were cultured in monolayer with EMD (1, 10, 50,100, 250μg/ml) in aMEM supplemented with 2% FBS for 3 days. Cells cultured in aMEM supplemented with 2% FBS (basal medium) served as the control group. Double-stranded DNA was quantified by PicoGreen assay. Quantitative RT-PCR was performed to determine the expression levels of RUNX2, osteopontin (OPN) and osteocalcin (OCN), dentin matrix protein1 (DMP1) and dentin sialophosphoprotein (DSPP) at different time points (day 0, 5 and 10) when exposed to 10μg/ml EMD or basal medium. Alkaline phosphatase specific activity (ALPSA) was determined after 5 and 10 days culture. Mineral deposition (as calcium) was visualised using Alizarin Red staining.
Results
All EMD groups showed increased dsDNA content after 3 days culture. However, EMD at 50μg/ml appeared optimal giving a 6.8 fold increase over the control group (p<0.01). Relative to day 0, RUNX2 expression was reduced in the presence of 10μg/ml EMD by 50% after 5 days culture (it was reduced by 30% in the control group), After 10 days culture, RUNX2 expression increased 1.5 fold in the presence of 10μg/ml EMD (increased 1.2 fold in the control group) OPN expression patterns were broadly similar to RUNX2. However, after 5 days culture OCN expression increased 4 fold (increased 7 fold in the control group) but by day 10, expression levels in the presence of EMD had fallen back to day 0 levels whereas levels in the control still showed a 6 fold increase compared to day 0. DMP1 and DSPP expression levels remained at very low levels over the full 10 days in culture. After 10 days culture, HBMSCs treated with 10μg/ml EMD showed significant increase of ALPSA compared with the control group. Mineral deposition was observed in both groups, but to a visibly greater extent in EMD treated groups.
Conclusions
EMD stimulated HBMCs proliferation and osteogenic differentiation in vitro, suggesting that EMD may be a useful adjunct in inducing HBMSCs down an osteogenic lineage for use in bone tissue engineering applications.
Acknowledgments
ORSAS, LDI Bursary, Xiros.
