Regulation of osteogenic differentiation of rat bone marrow stromal cells on 2D nanorod substrates

Bone marrow stromal cells (BMSCs) possess multi-lineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors. Although, they have shown great prospects in therapeutic and medical applications, less is known about their behavior on nanosurfaces mimicking the extra cellular matrix (ECM). In this report we have employed 2D substrates coated with tobacco mosaic virus (TMV) nanorods to study the differentiation process of BMSCs into osteoblast like cells. TMV is a rod-shaped plant virus with an average length of 300 nm and diameter of 18 nm. The osteogenic differentiation of BMSCs on TMV was studied over time points of 7, 14 and 21 days. We examined the temporal gene expression changes during these time points by real-time quantitative PCR (RT-qPCR) analysis. As expected, osteo-specific genes (osteocalcin, osteopontin and osteonectin) were upregulated and showed a maximum change in expression on TMV at 14 days which was 7 days earlier than on tissue culture plastic (TCP). Based on the genes expression profile generated by RT-qPCR experiments, we proposed that the early interaction of cells with TMV triggers on signaling pathways which regulate speedy expression of osteocalcin in turn, resulting in early mineralization of the cells. To further investigate these regulating factors we studied global changes in gene expression (DNA microarray analyses) during osteogenic differentiation on the nanosubstrate. Multitudes of genes were affected by culturing cells on nanorod substrate, which corroborated our initial PCR findings. Microarray analysis further revealed additional targets influenced by the presence of nanorods on the surface, of which, the expression of bone morphogenetic protein 2 (BMP2) was of particular interests. Further investigation into the temporal change of BMP2, revealed that it acts as a major promoter in signaling the early regulation of osteocalcin on TMV coated substrates.