The impact of nitric oxide on calcium homeostasis in PE/CA-PJ15 cells.

OBJECTIVE: Nitric oxide (NO) production and Ca(2+) homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca(2+) homeostasis in a highly complex and cell-specific manner and although Ca(2+) homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca(2+)]c homeostasis in PE/CA-PJ15 cells. DESIGN: Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca(2+) concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La(3+) were employed to perturb intracellular calcium levels. RESULTS: NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca(2+)]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca(2+) homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca(2+)]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca(2+)]c was also reduced in the presence of La(3+) or thapsigargin. CONCLUSIONS: NO induces a concentration-dependent [Ca(2+)]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca(2+) buffering in the presence of TG or La(3+). Further, we show that exogenous NO deregulates Ca(2+) homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.

Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats.

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Hydrogenated amorphous carbon nanopatterned film designs drive human bone marrow mesenchymal stem cell cytoskeleton architecture.

The interaction between stem cells and biomaterials with nanoscale topography represents a main route in the roadmap for tissue engineering-based strategies. In this study, we explored the interface between human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and hydrogenated amorphous carbon (a-C:H) film designed with uniform, groove, or grid nanopatterns. In either case, hBM-MSCs preserved growth rate and multi-differentiation properties, suggesting that the films were biocompatible and suitable for stem cell culture. hBM-MSCs responded to different nanopattern designs with specific changes of microtubule organization. In particular, the grid pattern induced a square-localized distribution of alpha-tubulin/actin fibers, whereas the groove pattern exerted a more dynamic effect, associated with microtubule alignment and elongation.