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In this study, we compared the efficiency of osteoblast differentiation media (ODM) containingthree distinct reagent combinations in osteoblastic differentiation of human bone marrowderivedmesenchymal stem cells (hBMSCs) in monolayer culture. In addition, we analyzedgrowth and differentiation of hBMSCs on silk scaffolds and examined the bone-forming activityof a nanofibrous silk scaffold in a tibia diaphysis defect model of a rat hind limb with intramedullarynailing. Although all three ODM increased alkaline phosphatase activity to a comparableextent, the ODM containing bone morphogenetic protein-2 (BMP-2) was found to be significantlyless effective in promoting mineral deposition than the others. Growth of hBMSCs onsponge-form silk scaffolds was faster than on nanofibrous ones, while osteoblastic differentiationwas apparent in the cells grown on either type of scaffold. By contrast, bone formationwas observed only at the edge of the nanofibrous scaffold implanted in the tibia diaphysisdefect, suggesting that use of the silk scaffold alone is not sufficient for the reconstitution of thelong bone defect. Since silk scaffolds can support cell growth and differentiation in vitro, loadingMSCs on scaffolds might be necessary to improve the bone-forming activity of the scaffoldin the long bone defect model.

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