Odontogenic differentiation of human dental pulp stem cells induced by preameloblast-derived factors

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.

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 [1], [2], [3]. 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 mechanisms occurring during the development of various organs, including hair follicles and mammary glands [5]. Tooth development is also achieved through continuous reciprocal interactions between the dental epithelium and the underlying ectomesenchyme. 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 differentiation 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 osteoblasts [7], [8]. In addition, hDPSCs interact with various biomaterials and are effective in mineralized tissue formation [9], [10], [11]. Therefore, hDPSCs would be an ideal material in clinical trials and tissue engineering for bone and dentin regeneration. However, dentin regeneration using adult pulp stem cells is limited by factors such as epithelial shortage, because the dental epithelium, including ameloblasts, degenerates, and apoptosis occurs after enamel formation in humans. In contrast, rodent incisors have a special epithelial structure, referred to as the ‘apical bud,’ at the apical end, which continuously grows [12]. The apical bud contains inner enamel epithelium (IEE) that differentiates into the ameloblasts, outer enamel epithelium, and stellate reticulum [13]. Therefore, murine apical bud cells (ABCs) can be used instead of the dental epithelium for the differentiation of odontoblasts and regeneration of dentin in humans.

Recently, proteomic methods are widely applied to cancer [14] and bacterial analysis [15]. Analysis of the conditioned medium (CM) of cultured cells from various diseases is ongoing [16]. Although proteomic analysis makes use of mineralized tissues, such as dentin [17] and enamel [18], secretory factors of the dental epithelium that are essential for the odontoblast differentiation have not yet been utilized for proteomic research.

In the present study, the effects of preameloblast-CM (PA-CM) from mouse ABCs on the odontogenic differentiation of hDPSCs were investigated in vitro and in vivo. Furthermore, we analyzed PA-CM by liquid chromatography-mass spectrometry (LC-MS/MS) to identify factors that facilitate odontoblast differentiation and dentin formation.