M Zhang1, Y-Q Bian, H-M Tao, X-F Yang, W-D Mu. 1. Department of Orthopedic, Liaocheng People's Hospital of Shandong, Liaocheng, China. cola.daughter@163.com.
Abstract
OBJECTIVE: This study was designed to investigate whether Simvastatin could facilitate osteogenic differentiation of rat marrow mesenchymal stem cells (MSCs) by modulating the Wnt/β-catenin pathway, thus promoting fracture healing. MATERIALS AND METHODS: MSCs were isolated from rat bone marrow specimens and their purity was identified. The third generation of MSCs was cultured in osteoinduction medium containing simvastatin of gradient concentration, and the highest dose of simvastatin that did not cause cell proliferation was determined by the result of the CCK8 assay. The effects of simvastatin on osteogenic differentiation of MSCs were evaluated by ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific gene expression. Finally, Wnt pathway antagonist DKK1 and β-catenin disturbing agent were added to MSCs to detect the ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific genes of MSCs respectively, and to evaluate whether simvastatin promoted osteogenic differentiation of MSCs by activating Wnt/β-catenin pathway. RESULTS: After osteoinduction, simvastatin of 0.3 nmol/L was found to be the highest dose that did not induce the proliferation of MSCs. After treated with 0.3 nmol/L simvastatin for 7 days, the ALP activity of cells and the number of cell calcified nodules significantly increased. Meanwhile, the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were clearly up-regulated. However, when the MSCs were treated with DKK1 for 7 days, the ALP activity and the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were found decreased. Simvastatin markedly up-regulated the expression of the β-catenin protein, while transfection of β-catenin shRNA inhibited the expression of osteoblast-related genes including ALP, Runx2, OCN, and OPN. CONCLUSIONS: Simvastatin can promote the differentiation of rat MSCs into osteoblast-like cells, and its mechanism may be related to the Wnt/β-catenin pathway.
OBJECTIVE: This study was designed to investigate whether Simvastatin could facilitate osteogenic differentiation of rat marrow mesenchymal stem cells (MSCs) by modulating the Wnt/β-catenin pathway, thus promoting fracture healing. MATERIALS AND METHODS: MSCs were isolated from rat bone marrow specimens and their purity was identified. The third generation of MSCs was cultured in osteoinduction medium containing simvastatin of gradient concentration, and the highest dose of simvastatin that did not cause cell proliferation was determined by the result of the CCK8 assay. The effects of simvastatin on osteogenic differentiation of MSCs were evaluated by ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific gene expression. Finally, Wnt pathway antagonist DKK1 and β-catenin disturbing agent were added to MSCs to detect the ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific genes of MSCs respectively, and to evaluate whether simvastatin promoted osteogenic differentiation of MSCs by activating Wnt/β-catenin pathway. RESULTS: After osteoinduction, simvastatin of 0.3 nmol/L was found to be the highest dose that did not induce the proliferation of MSCs. After treated with 0.3 nmol/L simvastatin for 7 days, the ALP activity of cells and the number of cell calcified nodules significantly increased. Meanwhile, the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were clearly up-regulated. However, when the MSCs were treated with DKK1 for 7 days, the ALP activity and the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were found decreased. Simvastatin markedly up-regulated the expression of the β-catenin protein, while transfection of β-catenin shRNA inhibited the expression of osteoblast-related genes including ALP, Runx2, OCN, and OPN. CONCLUSIONS:Simvastatin can promote the differentiation of rat MSCs into osteoblast-like cells, and its mechanism may be related to the Wnt/β-catenin pathway.
Authors: Antonio G B Castro; Dennis W P M Löwik; Mies J van Steenbergen; John A Jansen; Jeroen J J P van den Beucken; Fang Yang Journal: RSC Adv Date: 2018-08-10 Impact factor: 4.036