Abstract
Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Because the interest in carbon nanomaterials has grown within the last decade in light of a wide variety of applications, the aim of this study was to test and evaluate the suitability and cytocompatibility of a particular nanometer-thin nanocrystalline glass-like carbon film (NGLC) composed of curved graphene flakes joined by an amorphous carbon matrix. This material is a disordered structure with high transparency and electrical conductivity. For this purpose, we used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryos. Cells were cultured either in a powder of increasing concentrations of NGLC microflakes (82±37μm) in the medium or on top of nanometer-thin films bathed in the same culture medium. The metabolism activity of SN4741 cells in presence of NGLC was assessed using methylthiazolyldiphenyl-tetrazolium (MTT) and apoptosis/necrosis flow cytometry assay respectively. Growth and proliferation as well as senescence were demonstrated by western blot (WB) of proliferating cell nuclear antigen (PCNA), monoclonal phosphorylate Histone 3 (serine 10) (PH3) and SMP30 marker. Specific dopaminergic differentiation was confirmed by the WB analysis of tyrosine hydroxylase (TH). Cell maturation and neural capability were characterized using specific markers (SYP: synaptophysin and GIRK2: G-protein-regulated inward-rectifier potassium channel 2 protein) via immunofluorescence and coexistence measurements. The results demonstrated cell positive biocompatibility with different concentrations of NGLC. The cells underwent a process of adaptation of SN4741 cells to NGLC where their metabolism decreases. This process is related to a decrease of PH3 expression and significant increase SMP30 related to senescence processes. After 7 days, the cells increased the expression of TH and PCNA that is related to processes of DNA replication.On the other hand, cells cultured on top of the film showed axonal-like alignment, edge orientation, and network-like images after 7 days. Neuronal capability was demonstrated to a certain extent through the analysis of significant coexistence between SYP and GIRK2. Furthermore, we found a direct relationship between the thickness of the films and cell maturation. Although these findings share certain similarities to our previous findings with graphene oxide and its derivatives, this particular nanomaterial possesses the advantages of high conductivity and transparency. In conclusion, NGLC could represent a new platform for biomedical applications, such as for use in neural tissue engineering and biocompatible devices.
Highlights
Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells
We evaluate the capacity of nanocrystalline glass-like carbon film (NGLC) in the form of thin films (5–80 nm) and powder suspended in the cell culture medium for biocompatibility, which would promote normal cell growth, cell differentiation and cell maturation, for which, in the case of this new carbon-based biomaterial, no data have been reported to date
As was extensively described in our recent publication [15], we were able to define the thickness of these films, and their optical and electrical properties, by carefully controlling the gas flow ratios used in the chemical vapor deposition (CVD) procedure (Fig 1A and 1B)
Summary
Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Interest in carbon nanomaterials with high transparency and electrical conductivity has grown within the last decade in light of a wide variety of applications, including their use in biocompatible sensors, diagnostic devices and bioelectronic implants [1]. Among the second group, disordered structures, diamond-like carbon has been proposed as biocompatible and bioactive surface coatings that can promote and stabilize cell attachment [10], promotes the formation of functional neuronal networks [11] and can be use as a tailorable and tunable substrate to study neural cell biology in vitro and in vivo [13]
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