Wei Qin1, Xianling Gao1, Tao Ma2, Michael D Weir3, Jing Zou2, Bing Song3, Zhengmei Lin4, Abraham Schneider5, Hockin H K Xu6. 1. Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China; Department of Advanced Oral Sciences and Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland. 2. Department of Oncology and Diagnostic Sciences, University of Maryland School of Medicine, Baltimore, Maryland. 3. Department of Advanced Oral Sciences and Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland. 4. Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China. Electronic address: linzhm@mail.sysu.edu.cn. 5. Department of Oncology and Diagnostic Sciences, University of Maryland School of Medicine, Baltimore, Maryland. Electronic address: schneider66@umaryland.edu. 6. Department of Advanced Oral Sciences and Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland; Center for Stem Cell Biology and Regenerative Medicine, University of Maryland School of Medicine, Baltimore, Maryland; Department of Mechanical Engineering, University of Maryland, Baltimore County, Baltimore County, Maryland. Electronic address: hxu@umaryland.edu.
Abstract
INTRODUCTION: Metformin is a first-line drug for treating type 2 diabetes that regulates the differentiation of mesenchymal stem cells. Its effects on human dental pulp cells (DPCs) remain unknown. This study aimed to investigate the effects of metformin on the proliferation and differentiation of DPCs. METHODS: A live/dead viability assay kit was used to examine the effects of metformin on the cell viability of DPCs. Cell proliferation was analyzed using a cell counting kit (CCK-8; Dojindo, Tokyo, Japan). Levels of phosphorylated and unphosphorylated adenosine 5'-monophosphate-activated protein kinase (AMPK) were quantified by Western blot analysis in response to metformin and the AMPK signaling inhibitor Compound C (EMD Chemicals, San Diego, CA). The effects of Compound C on the metformin-induced odontoblast differentiation of DPCs were determined by alkaline phosphatase activity assay and von Kossa staining, and the expression of odontoblastic markers was evaluated by reverse-transcription polymerase chain reaction analysis. RESULTS: DPCs exhibited mesenchymal stem cell characteristics using flow cytometry. Different doses of metformin were shown to be cytocompatible with DPCs, yielding >90% cell viability. None of the concentrations of metformin up to 50 μmol/L affected cell proliferation. The Western blot assay showed that DPCs express functional organic cation transporter 1, a transmembrane protein that mediates the intracellular uptake of metformin. Metformin significantly activated the AMPK pathway in a dose-dependent manner. In addition, it stimulated alkaline phosphatase activity; enhanced mineralized nodule formation; and increased the expression of odontoblastic markers including dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osteocalcin. Moreover, pretreatment with Compound C, a specific AMPK inhibitor, markedly reversed metformin-induced odontoblastic differentiation and cell mineralization. CONCLUSIONS: This study shows that metformin can induce DPC differentiation and mineralization in an AMPK-dependent manner and that this well-tolerated antidiabetic drug has potential in regenerative endodontics as well as in other regenerative applications.
INTRODUCTION:Metformin is a first-line drug for treating type 2 diabetes that regulates the differentiation of mesenchymal stem cells. Its effects on human dental pulp cells (DPCs) remain unknown. This study aimed to investigate the effects of metformin on the proliferation and differentiation of DPCs. METHODS: A live/dead viability assay kit was used to examine the effects of metformin on the cell viability of DPCs. Cell proliferation was analyzed using a cell counting kit (CCK-8; Dojindo, Tokyo, Japan). Levels of phosphorylated and unphosphorylated adenosine 5'-monophosphate-activated protein kinase (AMPK) were quantified by Western blot analysis in response to metformin and the AMPK signaling inhibitor Compound C (EMD Chemicals, San Diego, CA). The effects of Compound C on the metformin-induced odontoblast differentiation of DPCs were determined by alkaline phosphatase activity assay and von Kossa staining, and the expression of odontoblastic markers was evaluated by reverse-transcription polymerase chain reaction analysis. RESULTS:DPCs exhibited mesenchymal stem cell characteristics using flow cytometry. Different doses of metformin were shown to be cytocompatible with DPCs, yielding >90% cell viability. None of the concentrations of metformin up to 50 μmol/L affected cell proliferation. The Western blot assay showed that DPCs express functional organic cation transporter 1, a transmembrane protein that mediates the intracellular uptake of metformin. Metformin significantly activated the AMPK pathway in a dose-dependent manner. In addition, it stimulated alkaline phosphatase activity; enhanced mineralized nodule formation; and increased the expression of odontoblastic markers including dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osteocalcin. Moreover, pretreatment with Compound C, a specific AMPK inhibitor, markedly reversed metformin-induced odontoblastic differentiation and cell mineralization. CONCLUSIONS: This study shows that metformin can induce DPC differentiation and mineralization in an AMPK-dependent manner and that this well-tolerated antidiabetic drug has potential in regenerative endodontics as well as in other regenerative applications.
Authors: S Gronthos; J Brahim; W Li; L W Fisher; N Cherman; A Boyde; P DenBesten; P Gehron Robey; S Shi Journal: J Dent Res Date: 2002-08 Impact factor: 6.116
Authors: Obi Egbuniwe; Bernadine D Idowu; Juan M Funes; Andrew D Grant; Tara Renton; Lucy Di Silvio Journal: Cell Cycle Date: 2011-11-15 Impact factor: 4.534