Augmenting osteogenesis with BMPs and miRNA-2887 in Canine MSCs

Abstract

Clinicians are currently still challenged with large bone defects and their treatment, due to absent or delayed vascular ingrowth, which causes hypoxic conditions at the fracture site. Several therapeutic strategies have been developed, involving growth factors, grafts, scaffolds and stem cells. Complications still exist in these therapies and therefore new therapy strategies are tested in vitro with the use of mesenchymal stem cells (MSC) that undergo osteogenic differentiation. Important inducers of osteogenesis are dexamethasone, a glucocorticoid, and bone morphogenetic proteins (BMP), which are growth factors. Dexamethasone shows to have a negative effect in human MSCs when given the entire culture period, due to inhibition of proliferation and decreased viability and therefore dexamethasone should only be added the first 7 days of culture. However, this study demonstrated that canine MSCs cultured with osteogenic medium during the entire culture period of 21 days reveal the best osteogenic differentiation when compared with culture with osteogenic medium without dexamethasone and osteogenic medium supplemented with dexamethasone only for the first 7 days. Under optimal osteogenic conditions, BMP-2 and BMP-6, the most potent inducers of all BMPs in rodents and human, were assessed for their additive effect on osteogenesis. Both in monolayers and aggregates, BMP-2 100ng/ml revealed to have the best osteogenic potential in comparison with BMP-6 and the highest noggin expression in aggregates. The greater potential of BMP-2 in inducing osteogenesis is in again in contrast with humans and rodents and could be caused by species-specific BMP-receptor expression differences and noggin. Noggin in rodents functions as an antagonist of BMPs, except for BMP-6. Therefore, BMP-6 is found to be more potent in mice and rats compared with other BMPs. In the final study performed, noggin was supressed using a microRNA (miRNA), a type of RNA involved in gene regulation, and it was expected that this would enhance osteogenesis as reported in rodents. miRNA-2887 at a concentration of 50nM was transfected in canine MSCs treated with osteogenic medium supplemented with BMP-2 100ng/ml and resulted in suppression of noggin. Surprisingly, noggin suppression did not lead to an increase of osteogenic markers, but a decrease. In conclusion, in canine MSCs BMP-2 was found to be more potent in augmenting osteogenesis compared with BMP-6, even though noggin was highest expressed in BMP-2 conditions, while suppression of noggin expression negatively affected osteogenesis. Altogether, this indicates that noggin functions in a similar fashion as in humans by agonizing BMPs.