Nanotopographical patterning of biomaterial substrates has great potential for biofunctionalisation of devices for clinical applications, such as in orthopaedics. Nanotopography comprising 120 nm diameter nanopits with a partially disordered arrangement of up to +/- 50 nm offset from a square lattice with 300 nm centre to centre spacing (NSQ50, fabricated by electron beam lithography) has been characterized as being osteogenic. Following the finding of osteogenesis of mesenchymal stromal cells derived from human bone marrow (MSCs) on the NSQ50 nanotopography, MSCs cultured on ε-polycaprolactone (PCL) embossed with the NSQ50 pattern was used for this study on molecular mechanisms underlying NSQ50 induced MSC osteogenesis: the functional coupling of gene expression and osteogenesis, the molecular regulatory events driving gene expression and osteogenesis, and the possible link of metabolomics with molecular signalling of MSCs on the NSQ50 surface. Temporal analysis of gene expression for MSCs on the NSQ50 surface revealed that MSC fate commitment and osteogenic differentiation was transcriptionally controlled. The cell cycle and growth regulating transcription factor C-MYC was found to be significantly repressed, whereas the osteogenic transcription factor RUNX2 was up-regulated at 5 days of cell culture, and this was followed upregulation of the osteoblast specific transcription factor osterix (OSX) at days 11 and 13. Following this transcription factor activation, osteoblast specific marker genes were induced with increased alkaline phosphatase (ALP) observed at day 16, increased osteopontin (OPN) at day 20 and increased osteocalcin (OCN) at day 28. These data suggested that transcription factors regulated MSC osteogenic commitment at the early stage, and induced osteogenic specific marker gene expression at the late stages of cell culture on the NSQ50 surface, resulting in osteogenesis of the MSCs. Signalling pathway analysis illustrated that bone morphogenetic protein 2 (BMP2) was the initial signalling molecule that triggered osteogenic differentiation of MSCs by inducing RUNX2 expression via the canonical SMAD pathway. BMP2 and its transmembrane receptor type 1A (BMPR1A) were stimulated by 3 nanotopographical cues by 3 days of cell culture on the NSQ50 surface, whereas the induction of other transmembrane receptors, including the low density lipoprotein-receptor related protein 5 (LRP5) and integrin subunits α3, α4, β1, and β3 were not observed. Inhibition of BMP2 signalling by the BMP2 antagonist noggin resulted in down-regulation of RUNX2 and ALP. Further analysis of BMP2 signalling revealed that BMP2 also modulated expression of the microRNA (miR)- 23b which targets RUNX2. The effect of BMP2 signalling on the expression of RUNX2 was enhanced by co-localizing with integrin αvβ5 (the vitronectin (VN) receptor) which was found to be up-regulated after 5 days cell culture. Metabolomics data for MSCs on the NSQ50 surface during early osteogenic differentiation was analysed. MSC cellular metabolite analysis revealed possible changes in bioenergetic balance with shifts towards more mitochondrial oxidative process, possibly indicating a switch in MSCs on the surface towards lineage-specific commitment. Further analysis of the metabolomics data illustrated PPARG ligands from the polyunsaturated fatty acid family was downregulated, suggesting the inhibition of adipocyte differentiation in MSCs on the surface. The down-regulation of unsaturated fatty acids could also be involved in the regulation of Ca2+ channels which positively regulate BMP2 expression. The metabolomics data, together with gene expression and signalling pathway analysis demonstrated that MSCs on the NSQ50 surface initiated osteogenic commitment after 3 days of cell culture, with BMP2 initiating osteogenic transcription factor stimulation of mature and functional osteoblasts on the surface.