백서 골수에서 분리, 배양한 간엽 간세포의 시험관내 골형성 및 생체내 골형성의 유도

Abstract

Objectives : To induce osteogenesis of cultured mesenchymal stem cells(MSCs) isolated from rat bone marrow in vitro and in vivo. Methods : Bone marrow cells were isolated from Spraugue-Darwley rats and seeded in culture medium. MSCs were isolated from bone marrow cells and cultured. For in vitro induction of osteogenesis, MSCs were cultured in osteogenic medium. After 2 weeks, evaluation of osteogenesis was performed with alkaline phosphatase (ALP) activity. ALP immunochemical stain, von-Kossa stain for mineral deposition, and Northern blot analysis for osteocalcin m-RNA. For in vivo induction of osteogenesis, MSCs were seeded on Collagraft strips. The Collagraft strips seeded with MSCs were grafted in the subcutaneous tissue of the back of immunocompromised rats. Grafted Collagraft strips were harvested 3, 6, 9, 12 weeks after graft and stained with H-E and toluidine blue-O stain. Results : In vitro assay ALP activity was 121.27 p-nitrophenol nmol/106cell in osteogenesis induced MSCs and 75.53 p-nitrophenol nmol/106cell in control group. Osteogenesis induced MSCs were strongly stained by ALP immunochemical stain. Von Kossa stain showed black mineral deposition. There were strong bands in Northern blot analysis for osteocalcin m-RNA. H-E stain showed penetration of cellular component into the pores of Collagraft strip in all specimen and showed formation of osteoid from 6 weeks after graft. Toluidine blue-O stain showed blue staining cartilaginous component from 3 weeks after grafting. Conclusion : We could induce osteogenesis from mesenchymal stem cells in vitro and in vivo. Based on the results of this study, bone graft might be able to replaced by a new method in the near future.