Regeneration of Dentin Using Stem Cells Present in the Pulp

*Toshiyuki Kawakami, Kiyofumi Takabatake, Hotaka Kawai, Keisuke Nakano, Hidetsugu Tsujigiwa and Hitoshi Nagatsuka*

#### **Abstract**

Dentin is one of the major hard tissues of the teeth. Dentin is similar to bone in texture, but it is different from bone tissue histologically. It is formed by odontoblasts; however, these cells are present in a limited area in the human body and are not found anywhere other than the dental pulp. It is difficult to collect and proliferate mature odontoblasts for regenerative medicine. However, odontoblast are necessary for regenerating dentin. It is known that odontoblasts differentiate from mesenchymal stem cells in the dental pulp during tooth development. Dentin can be generated using the stem cells present in the pulp. Many stem cells are recruited from the bone marrow to the teeth, and it is possible that the stem cells present in the pulp are also supplied from the bone marrow. Herein, we explain the mechanism of stem cell supply to the teeth and the possibility of dentin regeneration by specific cell differentiation induction methods.

**Keywords:** dental pulp cell, odontoblast, bone marrow-derived cell, mesenchymal stem cell, differentiation induction

### **1. Introduction**

Stem cells have the special property of differentiating into different types of cells. In the human body, stem cells are present in organs with a high regenerative and proliferative ability. Bone marrow is an organ that supplies blood cells throughout the body. The bone marrow is also rich in stem cells. The special nature of stem cells in the bone marrow is that they can migrate to all tissues throughout the body. Bone marrow-derived cells (BMDCs) that appear in peripheral tissues have the characteristics of stem cells and show multiple differentiation. Recently, many researchers have shown that BMDCs may differentiate into a variety of organs, including skeletal muscle, hepatocytes, neurons, myocardium, mucosal epithelial cells, and blood vessels [1, 2]. This is an important fact because such cells can be used to regenerate organs for treating various diseases [3]. The local supply and delivery of BMDCs has been extensively studied for treating ischemic diseases, including peripheral tissue ischemia and myocardial infarction, and has been attempted as a therapeutic tool [4, 5].

In previous studies, we used bone marrow transplant animal models in which bone marrow cells were transplanted from green fluorescent protein (GFP) transgenic mice to investigate the ability of BMDCs to be distributed and differentiated in the bones and teeth. GFP-positive cells were observed diffusely in the pulp and

periodontal ligament of mouse incisors, Langerhans cells in the oral epithelium, stromal fibroblasts, blood vessels, and osteoclasts in the tooth area [6]. Tissue stem cell differentiation is triggered by a variety of stimuli. We have shown previously that orthodontic mechanical stress and artificial inflammation stimulate the cells of the periodontium and dental pulp to express various cell differentiation factors. The osteoblast marker alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), which induces osteoblast differentiation, and heat shock proteins (HSPs), which are involved in homeostasis and cell differentiation, may be strongly expressed in cells exposed to injurious stimuli [7–12]. Moreover, periodontal tissue and dental pulp respond to mechanical stresses and inflammation, causing periodontal tissue remodeling and expression of cell differentiation-related factors [13]. Furthermore, orthodontic mechanical stress on the periodontium causes activation of hard tissue-forming cells in the pulp tissue [10, 11]. This indicates the potential of dental pulp stem cells to differentiate into hard tissue-forming cells upon stimulation. Moreover, mechanical stress and inflammation on the periodontal tissue affect the local recruitment of BMDCs [13, 14].

Recently, dental pulp has been proposed as a promising source of pluripotent mesenchymal stem cells for use in a variety of clinical applications. We isolated stem cells from the pulp, which is rich in BMDCs. The isolation and culture of dental pulp cells is an important factor. We attempted to establish cells that induce dentin-like hard tissue from the cells present in rat dental pulp and succeeded in establishing cells that resemble odontoblasts under *in vitro* conditions. These cells were named as the tooth matrix-forming GFP rat-derived cells (TGCs). The TGCs were maintained in cultures over 80 passages without showing any changes in morphology or properties. Physiological dentin has a characteristic structure, which is derived from the cell polarity of odontoblasts. However, the TGCs form dentinlike hard tissue under *in vivo* conditions but do not lead to the induction of polarized odontoblasts. Conversely, the geometric structure of biomaterials is considered important for inducing cell differentiation and tissue formation. Focusing on the importance of the geometry of artificial biomaterials in inducing cell differentiation and hard tissue formation, we have already succeeded in developing new honeycomb tricalcium phosphate (TCP) structures with holes of various diameters. We used the honeycomb TCP as a scaffold to induce TGCs into odontoblasts for the purpose of inducing odontoblasts with cell polarity [15].
