Background In the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene. Results In this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX). Conclusions Overall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering. Graphic abstract

Background Traditionally, severe bone defects caused by trauma or disease are treated with autologous bone grafts, but can result in additional pain and discomfort for the patient. Bone tissue engineering strategies involving the use of osteoprogenitor cells and novel scaffolds offer hope as an alternative therapy for the treatment of bone defects. We have demonstrated that adipose derived stem cells (ADSCs) grown on graphene nanoparticles with low oxygen content (LOG) results in spontaneous osteoblast differentiation in vitro. Due to the dark nature of the LOG surface, ADSCs cannot be monitored by traditional light microscopy. Hence, the goal of this work was to create fluorescent ADSCs that can be used for studies on LOG surfaces. Objective Determine if fluorescent ADSCs transduced by lentivirus undergo osteogenesis on low‐oxygen content graphene (LOG) surfaces. Hypothesis Fluorescently transduced ADSCs behave similarly to non‐transduced ADSCs on LOG coated surfaces. Methods Adipose tissue was obtained from human patients undergoing pannulectomies via an approved IRB and patient consent. In vitro expanded ADSCs were transduced at a multiplicity of infection (MOI) of 5 (>90% transduction efficiency) with lentivirus particles encoding green fluorescent protein (GFP), expanded in vitro, characterized by trilineage staining/flow cytometry and used for characterization on LOG surfaces. Results Our data show that ADSCs, obtained from two different human patients, undergo trilineage differentiation normally by histological staining and express positive surface markers (CD29, CD44, CD73, CD90, CD105) and lack expression of negative surface markers (CD34, CD45, CD106, HLA‐DR) indicative of stem cells, by flow cytometry. Moreover, these same ADSCs transduced by lentivirus particles, encoding GFP, undergo trilineage differentiation normally and expression of fluorescence is not reduced/lost upon differentiation. GFP expressing ADSCs seeded, grown and visualized on LOG coated surfaces demonstrated spontaneous osteogenesis in the absence of exogenous inducing factors compared to control ADSCs on polystyrene dishes via alizarin red staining and quantitation. Furthermore, addition of dexamethasone, beta‐glycerophosphate and ascorbic acid resulted in higher osteogenesis on LOG coated surfaces. Conclusions Based on these data we conclude the following: 1) lentivial transduction of ADSCs does not alter trilineage differentiation potential; 2) GFP expressing ADSCs can be used to continuously monitor cells on LOG coated surfaces; 3) Transduced ADSCs undergo osteogenesis similar to non‐transduced ADSCs on LOG coated surfaces; and 4) ADSCs obtained from different human patients behaved similarly on LOG coated surfaces suggesting LOG can be used for regenerative medicine purposes with any human ADSCs.

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.