Osteogenic Regulation by Graphene Nanoparticles in Human Mesenchymal Stem Cells

Abstract

Background Degenerative bone diseases caused by trauma, cancer, or aging imposes a global health care burden every year. Treatment strategies include directing stem cell differentiation through osteogenesis, thereby creating new bone material for repairment. We previously identified that adult mesenchymal stem cells (MSCs) cultured on graphene nanoparticles spontaneously supports osteoblast development in vitro. However, the osteo-genetic signaling of MSCs in the presence of graphene is largely unknown. Therefore, this study sought to measure genetic expressions of MSCs cultured on graphene using focused PCR arrays. Methods MSCs were extracted from human adipose tissue (hAD-MSCs) and human bone marrow (hBM-MSCs) and expanded in growth media. For experimental conditions, hAD-MSCs and hBM-MSCs were seeded on a graphene surface and cultured for either 7 or 21 days. At each time point, cells were extracted for total RNA and reverse transcribed to cDNA before loading onto RT2 Profiler PCR Human Osteogenesis Array (Qiagen, Hilden, Germany). Gene expressions of both cell lines were compared either over time (day 7 as the control to day 21 as the treated group) or between both cell lines at each time point (hAD-MSCs to hBM-MSCs at day 7 or day 21). Results were analyzed using Qiagen Gene Globe software to determine the relative fold change in comparison to the control. Fold changes were considered statistically significant at p < 0.05. Results We examined expression of four transcriptional genes known to control stem cell fate: DLX5, RUNX2, SOX9, and SP7. In hAD-MSCs, RUNX2 was up-regulated while SOX9 and SP7 were down-regulated, suggesting RUNX2 to be a master regulator in hAD-MSCs. In contrast, all genes were down-regulated in hBM-MSCs. However, when hBM-MSCs were compared to hAD-MSCs, all genes were up-regulated at both time points. Interestingly, the fold changes at day 7 were more robust compared to that observed at day 21, suggesting transcriptional regulation of osteogenesis in hBM-MSCs occurs at an earlier time point. Secondly, we examined common markers of osteogenesis including ALPL, BGLAP, PHEX, and SPP1. In hAD-MSCs, ALPL, BGLAP, and PHEX were upregulated, while SPP1 was down-regulated.In hBM-MSCs, ALP, PHEX, and SPP1 were up-regulated, overall suggesting osteo-genetic signaling by graphene occurs in both cell types. Conclusion hAD-MSCs and hBM-MSCs cultured on graphene showed significant changes in osteogenic gene expressions. These cells were cultured in absence of any chemical inducers, suggesting graphene nanoparticles alone commits osteo-genetic signaling of MSCs. We also observed that osteogenesis of hBM-MSCs may occur at an earlier time point than hAD-MSCs. Lastly, we identified DLX5 as a novel osteogenic regulator supported by graphene. Overall, we show that MSCs isolated from two independent tissue sources spontaneously express osteogenic genes in the presence of graphene.