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

Abstract

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.

Figure 1

(A) Schematic representation of different phases during osteogenic differentiation of BMSCs. Cells undergo early maturation and mineralization on TMV coated substrate as compared to flat TCP. (B) Representation of TMV wafer preparation by coating on 2D APTES wafers used in the study. The particle coverage on the wafer was characterized by AFM analysis (image on the right).

Figure 2

Adhesion of BMSCs on TMV coated wafers. (A) Representation of cell morphology after 2hrs (inset) and 24 hrs of incubation on TMV under serum free conditions. (B–D) SEM micrographs of BMSCs on TMV coated wafers. (B) shows TMV coated APTES wafer, fibrous structures observed (as marked by the arrow) could be due to bundled TMV particles visible on the wafer. (C) shows the interconnection cellular networks and (D) arrows show filopodia like extensions anchoring the cells on to the substrate. Scale bars are 100μm for (A), 10μm for (C) and 1 μm for (B and D).

Figure 3

(A) RT-qPCR analysis for the gene expression in the cells seeded on TMV and TCP under osteogenic conditions. For the cells seeded on TMV, the maximum up regulation in the osteocalcin (BGLAP) expression is observed around 14 days as compared to TCP where it achieves maximum up regulation by 21 days. In the graphs (**) represents p < 0.005 and (*) for p < 0.05 respectively. (B) Cells grown on TMV were analyzed for gene expression by immuno-staining for 7, 14 and 21 days (scale bar is 20 μm). (C) Cells aggregated to form sheet like structures around 14 days, which stained positive for BGLAP. The expression of SPP1 remained confined to the cells on the periphery of the sheets and neighboring cells. Scale bars are 20 μm for (B) and 100 μm for (C) i, iii and iv. Color representation: DAPI (blue), phalloidin (green), osteocalcin (BGLAP) (red), osteopontin (SPP1) (pink) and osteonectin (SPARC) (yellow).

Figure 4

(A) Network of osteogenic specific proteins regulated by BMP2 signaling. (B) RT-qPCR generated profile for the normalized expression of genes after 1 day of osteogenic induction on TMV coated substrates (normalized with respect to TCP, day 1). (C) Differential expression of the genes shown in the pathway calculated via gene array analyses.