| Objective:The study aimed to investigate the effect of macrophage secretion on the formation of microvascular-like structures of human umbilical vein endothelial cells(HUVECs)in the bone marrow mesenchymal stem cells(BMSCs)sheet,providing new ideas for vascularized tissue in tissue engineering.Methods:In this research,the resting state of macrophages(M0)were obtained by treating human myeloid leukemia mononuclear cells(THP-1)monocytes with 12-myristate 13-acetate(PMA)for 48h and rested for another 24h in culture medium without PMA.Differentiated the M0 into M1 phenotype in fresh medium supplemented with 20 ng/ml interferon-?(IFN-?)+100ng/ml lipopolysaccharide(LPS)and with 20ng/ml interleukin-4(IL-4)to the M2 phenotype.According the cell morphology and gene expression results by RT-PCR to characterize M0,polarized M1 and M2 cells.Culturing HUVECs,BMSCs cell sheets,HUVECs-BMSCs cell sheets in medium containing supernatant of M0,M1,M2 cells(CM)separately for 1,3,7 days,and in normal medium as control.The activity of those cells was measured by cell counting kit(CCK-8)and the effect of CM on cells'arrangement were observed under microscope.the HE staining was used to detect the thickness of cells sheets,the CD31immunohistochemistry and enzyme linked immunosorbent assays(Elisa)was used to access the microvascular network formation of the HUVECs-BMSCs sheets.Results:(1)After treating with PMA,THP-1 differentiated,extended pseudopodia and attached on plate.with the increase of PMA concentration,both 1×10~5/ml and2.5×10~5/ml THP-1 can differentiate into more M0 cells,but the 2.5×10~5/ml THP-1exhibited higher differentiation efficiency at high PMA concentration.(2)The macrophages'condition medium can enhance HUVECs'activity on Matrigel at day 1,but decreased cell activity at day 3 and 7.(3)the HUVECs in ECM emerged as colonies with a cobblestone-like morphology,the HUVECs in macrophages'condition medium exhibited spindle-shaped morphology and regularly in a certain direction rather than in disorder.With the extension of time,this performance becomes more obvious.(4)The activity of BMSCs cell sheets was lowest in CM(M1)at day 7,while it was higher in CM(M0)and CM(M2)than in the control group.(5)HE staining results showed that cell sheets of control group were thicker than that of CM,and the cells sheets of CM(M1)was the thinnest.(6)According to the results of CD31 immunofluorescence results,the BMSCs cell sheet acted as the scaffold for the migration of HUVECs and several HUVECs connected to form branches and vessel-like micron network structures.In the control group,the branches became more obvious over time.Although the branch network structures of HUVECs in each CM group differed depending on the state of the cell sheets,they were more obvious than in the control group.(7)In the experimental group of pre-vascularized BMSCs cell sheets,the Elisa results showed that the content of tumor necrosis factor-alpha(TNF-?)in CM(M1)was higher than in other CM groups at each time point,the content of vascular endothelial growth factor(VEGF)in the CM groups was higher than that in the control group,and the content of VEGF in CM(M1)was highest one,the content of platelet-derived growth factor-BB(PDGF-BB)was higher in CM(M2)compared with other groups.Conclusion:By paracrine,macrophage can regulate the viability and thickness of cell sheets and direct endothelial cells arrangement,as well as the content of angiogenic-related cytokines in the environment,ultimately affect the formation of microvascular structure in pre-vascularized BMSCs cell membrane. |
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