Influences of immunosuppressants on the osteogenic activity of rat mesenchymal stem cells in vitro & in vivo.

Abstract

Abstract

Influences of immunosuppressants on the osteogenic activity of rat mesenchymal stem cells in vitro & in vivo.

Yukyung Byun

Program in Periodontology, Department of Dental Science Graduate School, Seoul National University (Directed by Professor Yong-Moo Lee, D.D.S., Ph.D.)

Purpose The purpose of this study was to evaluate the influences of immunosuppressants on the proliferation and osteogenic differentiation of undifferentiated rat mesenchymal stem cells (MSCs) in vitro and bone regeneration of MSCs grafted rat calvarial defects in vivo. Methods MSCs obtained from rat femurs were cultured with immunosuppressants, FK506 and cyclosporine A (CsA), 50 and 500 nM respectively. Cell proliferations during 7 days were assessed with MTT assay. For osteogenic differentiation evaluation, alkaline phosphatase (ALP) analysis and alizarin red S staining were done. RT-PCR was used for the analysis of expression of mRNA for bone-related proteins. For in vivo study, MSCs were obtained from F344 rats and seeded in collagen matrixes. Animals for experiment were divided to 5 groups

control group (defect left without any treatment), Collagen group (defect filled with collagen matrix only), MSC group (defect filled with MSC-seeding matrix), FK506 group (MSC-seeding matrix was grafted and FK506 was injected for 4 weeks), and Rapamycin group (MSC-seeding matrix was grafted and rapamycin was injected for 4 weeks). At 4 and 8 weeks after surgery, the animals were sacrificed and histologic/micro-CT analysis was done. Results Cell proliferation was promoted more in FK506 groups than control or CsA groups. During the experimental period, FK506 groups showed increased ALP activity compared to other groups. Mineralization nodule formations were prominent in FK506 groups than control or CsA groups. Genes for osteopontin, osteonectin, and type I collagen were expressed more in FK506 groups, however the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulate on day 7 in FK506 groups. In 500 nM CsA group, most of the genes were less expressed compared to control. In histologic view of rat calvarial defects, FK506 group showed a prominent new bone formation. 3D-images analysis revealed that the defect fill of FK506 group was outstanding. The new bone area (%), bone mineral density, and bone volume/tissue volume (%) were greater in FK506 group than other groups. Rapamycin group did not show apparent new bone formation. Conclusions These results suggest that FK506 could enhance the osteogenic differentiation of rat MSCs and stimulate osteogenic activity of rat MSCs in rat calvarial defects. Other immunosuppressants show inconsistent results in vitro and in vivo.

Keywords: cell differentiation, cyclosporin A, FK506, rapamycin, immunosuppressants, rat mesenchymal stem cells Student number: 2007-30598