Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.

Perfusao antiblástica hipertérmica regional no melanoma de membros em adultos / Regional hyperthermic antiblastic perfusion in extremities melanoma in adults

The first antiblastic regional perfusion was performed in 1957, in New Orleans, to treat a patient with locally advanced melanoma. This technique allows to use high doses of antiblastic drugs and was improved about 10 years later by additional hyperthermia, an idea suggested by Cavaliere et al and by Stehlin. Three types of isolated perfusion are possible: normothermic antiblastic perfusion. The use of adjuvant hyperthermic antiblastic perfusion offers to patients with extremity melanomas an excellent chance of cure. To patients with ® in transit ¼ metastases, hyperthermic antiblastic perfusion offers the most effective treatment with limb salvage. The main complication in isolated perfusion is the escape of the perfusate to the systemic circulation, but methods for escape evaluation are improving with the utilization of radiolabelled serum albumin and gamma-detecting probes. The hyperthermia is one of the most promising means of cancer therapy and it can be a local, regional or systemic treatment. Several retrospective and a few prospective and randomized studies have demonstrated the superiority of using hyperthermic antiblastic perfusion in extremity melanoma as compared to other forms of treatment. The World Health Organization and the European Organization for Research and Treatment of Cancer are performing randomized and controlled studies to evaluate the role of hyperthermic antiblastic perfusion in melanoma