Odontogenic differentiation of human dental pulp stem cells induced by preameloblast-derived factors Ji-Hyun Lee a , Dong-Seol Lee a , Han-Wool Choung a , Won-Jun Shon b , Byoung-Moo Seo c , Eun-Hyang Lee d , Je-Yoel Cho d , Joo-Cheol Park a, * a Department of Oral Histology-Developmental Biology & Dental Research Institute, BK21 Project, School of Dentistry, Seoul National University, 28 Yeongun-dong, Chongro-gu, Seoul 110-749, Republic of Korea b Department of Conservative Dentistry, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea c Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea d Department of Biochemistry, School of Dentistry, Kyungpook National University and ProtAnBio Co., Ltd, Dae-gu 700-412, Republic of Korea article info Article history: Received 22 August 2011 Accepted 1 September 2011 Available online 16 September 2011 Keywords: Epithelial-mesenchymal interaction Preameloblast-CM Human dental pulp stem cell Odontoblast differentiation Dentin regeneration abstract The differentiation of odontoblasts is initiated by the organization of differentiating ameloblasts during tooth formation. However, the exact roles of ameloblast-derived factors in odontoblast differentiation have not yet been characterized. We investigated the effects of preameloblast-conditioned medium (PA- CM) on the odontogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. Furthermore, we analyzed the PA-CM by liquid chromatography-mass spectrometry to identify novel factors that facilitate odontoblast differentiation. In the co-culture of MDPC-23 cells or hDPSCs with mouse apical bud cells (ABCs), ABCs promoted differentiation of odontoblastic MDPC-23 cells and facilitated odontoblast differentiation of hDPSCs. PA-CM, CM from ABCs after 3 days culture, was most effective in increasing the dentin sialophosphoprotein promoter activity of odontoblastic MDPC-23 cells. When PA-CM-treated hDPSCs were transplanted into immunocompromised mice, they generated pulp- like structures lined with human odontoblast-like cells showing typical odontoblast processes. However, during recombinant human bone morphogenenetic protein 2-treated hDPSCs transplantation, some of the cells were entrapped in mineralized matrix possessing osteocyte characteristics. After proteomic analyses, we identified 113 types of proteins in PA-CM, of which we characterized 23. The results show that preameloblast-derived factors induce the odontogenic differentiation of hDPSCs and promote dentin formation. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Dentin forms the bulk of the tooth. Defects in dentin are common due to numerous pathologies, such as dental caries, mechanical trauma, or even genetic alterations. In the last decade, great progress in tooth regeneration including regeneration of dentin was made. However, strategies for dentin repair are mainly based on various growth factors, transcription factors, basement membrane components, and pulp-capping materials, such as calcium hydroxide or mineral trioxide aggregate [1e3]. Although these procedures may result in gains in pathologic reparative dentin formation, careful histological evaluation has indicated that none can fully restore the physiological architecture of the original dentin [4]. Thus, to achieve complete tissue regeneration, it is necessary to recapitulate the process involved in the original formation of the dentin during tooth development. Epithelial-mesenchymal interactions are important mecha- nisms occurring during the development of various organs, including hair follicles and mammary glands [5]. Tooth develop- ment is also achieved through continuous reciprocal interactions between the dental epithelium and the underlying ectomesen- chyme. Induction of ameloblasts derived from dental epithelial cells is indispensible for the differentiation of odontoblasts from ectomesenchymal cells during crown formation [6]. However, the exact roles of ameloblast-derived factors in odontoblast differen- tiation have not yet been characterized. Human dental pulp stem cells (hDPSCs) are easy to isolate from human third molars, are multipotent, and express mesenchymal stem cell markers. Human DPSCs can differentiate into various tissues, such as odontoblasts, adipocytes, chondrocytes, and * Corresponding author. Tel.: þ82 2 740 8668; fax: þ82 2 763 3613. E-mail address: jcapark@snu.ac.kr (J.-C. Park). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.09.007 Biomaterials 32 (2011) 9696e9706