Umemura Naoki, Ohkoshi Emika, Tajima Masamichi, Kikuchi Hirotaka, Katayama Tadashi, Sakagami Hiroshi
Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama, 350-0283, Japan.
Department of Oral Biochemistry, Asahi University School of Dentistry, Gifu, 501-0296, Japan.
Stem Cell Res Ther. 2016 Sep 20;7(1):135. doi: 10.1186/s13287-016-0399-8.
Dental pulp tissue contains many undifferentiated mesenchymal cells, which retain the ability to differentiate into mature cells. Induced pluripotent stem cells have been developed from various cell sources, including dental pulp-derived stem cells, and evaluated for potential application to regenerative therapy. Dental pulp tissues overexpress CD44, a cell-adhesion factor involved in the induction of mineralization. In this study, we investigated the effects of hyaluronan-a known CD44 ligand-on dental pulp stem cells (DPSCs).
DPSC CD44 expression was analyzed using immunofluorescence staining, flow cytometry, and western blotting. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Effects of hyaluronan on the cell cycle were analyzed by flow cytometry. Alkaline phosphatase activity was employed as marker of mineralization and measured by fluorometric quantification and western blotting. Bone morphogenetic protein (BMP)-2, BMP-4, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP-1) levels were measured using real-time polymerase chain reaction. Odontoblastic differentiation and the close cell signaling examination of DPSC differentiation were determined using western blotting.
Hyaluronan induced expression of the odontoblastic differentiation markers DMP-1 and DSPP. Moreover, the odontoblastic differentiation induced by hyaluronan was mediated by CD44-but not by Akt, Smad1 or MAPK signaling.
Our results indicate that hyaluronan induces odontoblastic differentiation of DPSCs via CD44. This suggests that hyaluronan plays a crucial role in the induction of odontoblastic differentiation from DPSCs. Our findings may aid the development of new, inexpensive, and effective conservative treatments for dental pulp repair.
牙髓组织含有许多未分化的间充质细胞，这些细胞保留了分化为成熟细胞的能力。诱导多能干细胞已从包括牙髓来源的干细胞在内的各种细胞来源中开发出来，并对其在再生治疗中的潜在应用进行了评估。牙髓组织过度表达CD44，这是一种参与矿化诱导的细胞粘附因子。在本研究中，我们研究了透明质酸（一种已知的CD44配体）对牙髓干细胞（DPSC）的影响。
使用免疫荧光染色、流式细胞术和蛋白质印迹法分析DPSC的CD44表达。使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法评估细胞增殖。通过流式细胞术分析透明质酸对细胞周期的影响。使用荧光定量法和蛋白质印迹法测量碱性磷酸酶活性作为矿化的标志物。使用实时聚合酶链反应测量骨形态发生蛋白（BMP）-2、BMP-4、牙本质涎磷蛋白（DSPP）和牙本质基质酸性磷酸蛋白1（DMP-1）的水平。使用蛋白质印迹法确定成牙本质细胞分化以及DPSC分化的紧密细胞信号检查。
透明质酸诱导成牙本质细胞分化标志物DMP-1和DSPP的表达。此外，透明质酸诱导的成牙本质细胞分化是由CD44介导的，而不是由Akt、Smad1或MAPK信号传导介导的。
我们的结果表明，透明质酸通过CD44诱导DPSC的成牙本质细胞分化。这表明透明质酸在诱导DPSC的成牙本质细胞分化中起关键作用。我们的发现可能有助于开发新的、廉价且有效的牙髓修复保守治疗方法。
