ABSTRACT
Introduction: Since photobiomodulation therapy (PBMT) favors in vitro mesenchymal stem cell (MSC) preconditioning before MSC transplantation, increasing the proliferation of these cells without molecular injuries by conserving their characteristics, in the present in vitro study we analyzed the effect of PBMT on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs). Methods: Irradiation with an InGaAIP Laser (660 nm, 10 mW, 2.5 J/cm2 , 0.08 cm2 spot size, and 10 s) was carried out. The cells were divided into four groups: CONTROL [cells grown in Dulbecco's Modified Eagle Medium (DMEM)], OSTEO (cells grown in an osteogenic medium); PBMT (cells grown in DMEM+PBMT), and OSTEO+PBMT (cells grown in an osteogenic medium plus PBMT). The cell proliferation curve was obtained over periods of 24, 48 and 72 hours using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Osteogenic differentiation was analyzed by the formation of calcium nodules over periods of 7, 14 and 21 days. Morphometric analysis was performed to quantify the total area of nodular calcification. Results: The highest cell proliferation and cell differentiation occurred in the OSTEO+PBMT group, followed by the PBMT, OSTEO and CONTROL groups respectively, at the observed times (P <0.05). Conclusion: PBMT enhanced the osteogenic proliferation and the differentiation of hUCMSCs during the periods tested, without causing damage to the cells and preserving their specific characteristics, a fact that may represent an innovative pretreatment in the application of stem cells.
ABSTRACT
A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.
