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  • Finally we seeded both cells isolated using the direct

    2018-10-29

    Finally, we seeded both SB431542 cost isolated using the direct attachment and the time-gradient attachment for 72h respectively into PLGA porous scaffolds and transplanted both constructs with cells respectively into the calvarial defects of rat to analyze their xenogenic reconstruction potentials. At 20weeks after transplantation, a significant bone formation was observed in areas grafted by both constructs with cells isolated using the direct attachment and the time-gradient attachment for 72h. The thickness of new bone formed in the graft area was thinner than that of rat calvaria surrounding the graft area, but the new bone in the graft area was relatively compact. As gross view and histological assay above, the construct with cells isolated using the time-gradient attachment for 72h seemed to have a superior ability of bone reconstruction compared to the construct with cells isolated using the direct attachment. According to histological assay, the new bone formation in the area grafted by the construct with cells isolated using the time-gradient attachment for 72h was superior to that in the area grafted by the construct with cells isolated using the direct attachment. It was confirmed by the statistical analysis of bone formation proportion. This inference should also be confirmed by the immunohistochemistry assay of human osteocalcin. The positive staining rate of osteocalcin from human in the area grafted by the construct with cells isolated using the time-gradient attachment for 72h was higher than that in the area grafted by the construct with cells isolated using the direct attachment. The immunogenicity of allogenic cells in the recipient is an important care of clinical cell transplantation. Our results showed that there is difference of immunogenicity and special immune modulation between both cells isolated using the time-gradient attachment for 72h and the direct attachment. Nevertheless, Poncelet et al. (2007) found that xenogenic MSCs with low immunogenicity in vitro elicited an in vivo immune response after transplantation. In our experiments, the inflammatory reaction, such as production of macrophages, was not observed in the area grafted by the construct with cells isolated using the time-gradient attachment for 72h. However, this reaction was elicited in the area grafted by the construct with cells isolated using the direct attachment. Though our result is not enough to prove the presentation of a local immune response, it may be an indication that a local inflammation occurred. The effect of PLGA scaffold on the inflammatory reaction was excluded since the same reaction had not been elicited in the area grafted only PLGA scaffold. Therefore, it is deduced that the immunogenic characteristics of cells isolated using the time-gradient attachment for 72h should be different from those of cells isolated using the direct attachment, i.e. cells isolated using the direct attachment might contain some other types of cells except for PMSCs. However, the in vivo immunogenicity of PMSCs isolated using the time-gradient attachment for 72h as a population of allogeneic cells and the in vivo inflammatory reaction of placenta-derived cells isolated using the direct attachment should be remained for further studies. In summary, PMSCs are isolated from human term placenta using the time-gradient attachment for 72h. In comparison with cells isolated using the direct attachment method, PMSCs isolated using the time-gradient attachment for 72h have the MSC-specific phenotype characteristic, the multi-differentiation potentials, the immunomodulation properties, and the higher xenogenic reconstruction potential. Therefore, the cells isolated using the time-gradient attachment for 72h should be promising for experimental and clinical applications.
    Materials and methods
    Introduction Embryonic stem (ES) cells are derived from cells of the inner cell mass at the blastocyst stage of embryogenesis and are capable contributing to the development of the organism through multiple rounds of division followed by specifically-timed programmed differentiation. In culture, murine ES cells can be characterized as having a rapid cell cycle, defined by short gap phases with a large proportion of the cell cycle devoted to S-phase (White and Dalton, 2005). This observation implies that a majority of gene products involved in both DNA replication and DNA repair should be active and readily accessible in these cells. Furthermore, the promoters of many of the genes involved in DNA replication contain E2F binding sites, and their expression is coordinately expressed upon entry into S-phase.