RESUMO
Objective To understand the differences in engulfing ability and phagolysosome for-mation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection. Methods Human THP-1 monocytes and HL-60 cells were pretreated with PMA ( phorbol-12-myristate-13-acetate) and ATRA ( all-trans retinoic acid) to differentiate them into mononuclear-macrophages and neutrophils, respec-tively. The phagocytosis of Leptospira interrogans in THP-1-PMA mononuclear-macrophages and HL-60-AT-RA neutrophils was detected by confocal microscopy. The morphology of intracellular Leptospira was deter-mined by transmission electron microscopy. The viability of phagocytized Leptospira and the percentages of dead Leptospira were analyzed by confocal microscopy and spectrofluorimetry, respectively. Confocal micros-copy was used to measure the formation of phagolysosomes in different phagocytes. Results Both THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils could phagocytize Leptospira interrogans, but the phagocytic ability of the former was notably stronger than that of the latter (P<0. 05). Intracellular Lep-tospira were surrounded by phagocytic vesicles in both types of phagocytes. THP-1-PMA mononuclear-mac-rophages were better than HL-60-ATRA neutrophils in killing intracellular Leptospira (P<0. 05). More phagolysosomes were formed in THP-1-PMA mononuclear-macrophages than in HL-60-ATRA neutrophils ( P<0. 05). Conclusions Human mononuclear-macrophages but not neutrophils act as major phagocytes that play an important role in phagocytizing and killing Leptospira during infection. Less fusion of the phagosomes with lysosomes may be responsible for the lower Leptospira-killing ability of neutrophils.
RESUMO
Objective: To investigate the effect of human tooth bone graft materials on the proliferation, differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods: Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials, OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder (dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A (DMEM medium containing 10% fetal bovine serum), group B (human tooth bone graft materials), group C (OSTEONⅡ synthetic highly resorbable bone grafting materials), group D (untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α (TNF-α ) and interleukin 6 (IL-6) expressions were detected by ELISA. Results: Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure. Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture, and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point ( P<0.05), but no significant difference was found among groups A, B, and C ( P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D ( P<0.05), but no significant difference was found among groups A, B, and C ( P<0.05). Conclusion: The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.