Abstract
Purpose
The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro.
Method
MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time quantitative PCR on day 14. Also assessed was alkaline phosphatase (ALP) activity by a colorimetric kinetic assay and Fast Blue B staining on day 14. Calcium deposition was determined by the calcium assay on day 35.
Results
The expression levels of integrin binding sialoprotein (IBSP) and osteocalcin (OC) were significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT and control groups (all p<0.038). Although the expression level of runt-related transcription factor 2 (RUNX2) was also reduced in NOGsi1 and NOGsi2 groups compared with NT and control groups, it did not reach statistical significance. ALP activity was significantly lower in NOGsi1 and NOGsi2 groups than that of NT group (both p<0.024). The same pattern was also observed in ALP Fast Blue B staining. Calcium deposition was also significantly decreased in both NOGsi1 and NOGsi2 groups compared with NT group (both p<=0.048).
Conclusion
Noggin suppression by siRNA inhibits BMP-2-induced osteogenesis of human bone marrow-derived MSCs. Our results, contrary to the extensive studies conducted in rodent cells/models, corroborated with the previous study that the addition of noggin in the cell culture increased osteogenesis of human MSCs. This suggests that the effects of noggin on BMP-2-induced osteogenesis of MSCs might be species-specific.
