Sasidharan et al. [71] reported that there was no LDH leakage of Vero cells GS-4997 treated with both pristine and functionalized graphene at different concentrations until 300 μg/mL. Recently, Zhang et al. [72] reported that cell cytotoxicity of dispersed nanographene platelets (NGPs) exhibited dose-dependent characters, which had no obvious cytotoxic effects to MG63 cells at a concentration A-1210477 less than 10 μg/mL, whereas it could delay cell cycle, promote cell apoptosis, damage cell microstructure, induce serious tumor necrosis factor-a expression, and greatly reduce ALP activity of MG63 cells at higher concentrations of NGPs. Zhang et al. [63] also reported that a few-layer graphene increased intracellular
generation of ROS and induced mitochondrial injury in neural cells after 4 and 24 h at a dose of 10 μg/mL. In contrast, surface-modified graphene and carboxylated graphene were reported to be less toxic than GO or native graphene [73, 74]. Figure 9 Effect of GO and S-rGO on LDH leakage in PMEF cells. LDH leakage was measured by changes in optical densities due to NAD+ reduction which were monitored at 490 nm, as described in Trichostatin A the ‘Methods’ section, using cytotoxicity detection lactate dehydrogenase kit. The results represent the means of three separate experiments, and error bars
represent the standard error of the mean. GO-treated groups showed statistically significant differences from the control group by Student’s t test (p < 0.05). Impact of GO and Branched chain aminotransferase S-rGO on ALP activity ALP activity is an important and quantitative marker of osteogenesis. Furthermore, ALP is an important marker for functional activity of cells such as cell proliferation. Cell numbers and ALP activity were used as measures of cell proliferation, self-renewal, and pluripotency. ALP is a membrane-bound enzyme that exhibits biphasic behavior. It is expressed
on the surface of pluripotent undifferentiated ES cells and disappears as cells begin to differentiate. To examine cell differentiation, the ALP was measured as a marker of differentiation. The ALP activity was measured in GO- and S-RGO-treated cells, and the results are represented in Figure 10. Alkaline phosphatase activity was quantified by hydrolysis of p-nitrophenyl phosphate after 4 days of treatment. As expected, GO-treated cells showed a dose-dependent decrease of the alkaline phosphatase activity. The addition of S-rGO significantly enhanced the alkaline phosphatase activity above that of the control or GO-treated groups. Aoki et al. [75] showed significant cell proliferation and ALP activity in single- and multiwall carbon nanotube (CNT)-treated SaoS2 cells, and they suggest that due to the structure and affinity of CNTs toward proteins, CNTs could be the potential scaffold material for tissue engineering. Zhang et al. [72] demonstrated that cells cultured with NGPs at low concentrations have a higher ALP expression close to the negative control group.