**2. Recruitment of BMDCs to periodontal tissue and dental pulp**

Caries are a major cause of pulpitis. When the tooth crown is destroyed by caries, the pulp cavity is perforated. High proliferative activity of the pulpal tissue results in chronic inflammatory hyperplasia [16]. Granulation tissue grows from the pulp and forms periodontal polyps that can grow from inside the pulp cavity to outside the pulp. Experimental histopathological studies have long been performed on periodontal polyps, including histological analysis and treatment [17–20]. However, there is a lack of knowledge about the origin of the cells present in the pulp. As a result, using an experimental system of GFP mouse bone marrow transplantation, this study revealed that the cells were derived from bone marrow mesenchymal cells. Our research group used an experimental system of GFP bone marrow transplanted mice to study the migration and differentiation of cells in different parts of the oral cavity and teeth. Muraoka et al. showed that the BMDCs migrate to periodontal tissue and differentiate into periodontal ligament cells, such as macrophages and osteoclasts [21]. Tomida et al. showed the pluripotency of BMDCs, which migrated to periodontium, after the application of orthodontic mechanical

#### *Regeneration of Dentin Using Stem Cells Present in the Pulp DOI: http://dx.doi.org/10.5772/intechopen.95589*

stress loading [13]. Kaneko et al. also reported the differentiation of BMDCs into cell components of periodontal tissue [14]. In our study, the method suggested by Osuga et al. [22] was used for the formation of granulation tissue through chronic inflammation in the dental pulp of GFP bone marrow transplanted mice [16]. Observations by micro-computed tomography (m-CT) [23, 24], histopathology, and immunohistochemistry were followed over time. Immunohistochemistry revealed notable results. Oka et al. used rats to observe periodontal tissue reactions in the tooth roots associated with dental pulp perforation [17]. In a similar study, Imaizumi et al. examined the spread of inflammatory lesions in the periodontal ligament [18]. Other experiments have shown the formation of inflammatory lesions in the pulp because of perforation of the pulp cavity, examining the types of cells that appear with inflammation and the evolution of the inflammatory state [19, 20]. A detailed histopathological examination by Nakamura et al. showed continuous granulation tissue growth in the pulp [25]. Thus, periodontal polyps are considered suitable for observing cell dynamics in the regeneration and repair of dental pulp tissue. The focus of previous studies was on histopathological examination, and the origin of the cellular components of pulp granulation tissue was not mentioned. Recently, it has been widely reported that mesenchymal stem cells derived from bone marrow migrate to various organs and play a role in tissue formation. To histologically examine the *in vivo* recruitment of bone marrow-derived undifferentiated mesenchymal cells, these cells need to be marked. GFP transgenic mice express GFP in all cells of the body. Therefore, in wild-type mice transplanted with GFP mouse bone marrow, it is possible to trace BMDCs using GFP as a marker [26, 27]. We used an experimental system using GFP bone marrow transplanted mice to perforate the pulp cavity of the maxillary left first molar and induce pulpitis. With the development of pulpitis, the origin of the cellular components involved in the growth of periodontal polyps was investigated.

The periodontal polyp model used in our experiment was based on Osuga's method [22]. Anesthetized GFP bone marrow transplanted mice were secured to the plate, and a hole was made in the crown of the maxillary left first molar using a dental cutting device. Thereafter, the pulp was histologically observed over time for 2 weeks, 1 month, 3 months, and 6 months.

At 2 weeks, granulation tissue proliferation with neutrophil infiltration occurred just below the pulpal perforation. Round to short oval cells appeared around the granulation tissue. The granulation tissue was composed of fibroblast-like cells, capillaries, and chronic inflammatory cells (**Figure 1**). Immunohistochemical examination of GFP revealed that GFP-positive cells made up the majority of the small oval cells that emerged around the granulation tissue. A small number of fibroblasts also showed a GFP-positive reaction (**Figure 1-d**).

At 1 month, the granulation tissue that proliferated in the pulp cavity increased. The number of short oval cells increased around the granulation tissue, and the number of fibroblasts and capillaries, which are the cellular components that make up the granulation tissue, also increased (**Figure 2**). Compared to the tissue at 2 weeks, the total number of GFP-positive cells increased. Most of the GFP-positive reactions occurred in small oval cells. However, the number of positive reactions in fibroblasts also increased (**Figure 2-d**).

At 3 months, the number of fibroblasts in the granulation tissue increased the most, and collagen fiber proliferation was also apparent. The number of capillaries also increased the most. Conversely, the number of small oval cells decreased (**Figure 3**). The number of GFP-positive cells in the tissue was the highest, with positive reactions occurring in small oval cells and fibroblasts (**Figure 3-d**).

At 6 months, the continuous growth of granulation tissue resulted in an increase in mature fibroblasts and collagen fibers. However, inflammatory cells, capillaries,

#### **Figure 1.**

*2-week-specimen. a: m\_CT image; b: Histopathological view of the same part of a, Scale bar = 200* μ*m; c: Enlarged view of b, Scale bar = 100* μ*m; d: IHC for GFP, Scale bar = 100* μ*m.*

#### **Figure 2.**

*1-month-specimen. A: m\_CT image; b: Histopathological view of the same part of a, scale bar = 200* μ*m; c: Enlarged view of b, scale bar = 100* μ*m; d: IHC for GFP, scale bar = 100* μ*m.*

#### **Figure 3.**

*3-month-specimen. A: m\_CT image; b: Histopathological view of the same part of a, scale bar = 200* μ*m; c: Enlarged view of b, scale bar = 100* μ*m; d: IHC for GFP, scale bar = 100* μ*m.*

#### **Figure 4.**

*6-month-specimen. A: m\_CT image; b: Histopathological view of the same part of a, scale bar = 200* μ*m; c: Enlarged view of b, scale bar = 100* μ*m; d: IHC for GFP, scale bar = 100* μ*m.*

and small oval cells decreased (**Figure 4**). The number of GFP-positive cells was reduced compared to that in the tissue at 3 months. However, many GFP-positive reactions appeared in the cells of mature granulation tissue.

A large number of GFP-positive cells appeared in periodontal polyps. Most of the positive cells were small oval cells. Over time, the number of GFP-positive fibroblasts increased. Therefore, these GFP-positive cells were derived from transplanted bone marrow cells. These GFP-positive cells were undifferentiated mesenchymal cells that migrated from the bone marrow to the pulp. From this, it was shown that the pulp has the potential to supply a large amount of BMDCs.
