**3. Cell differentiation of BMDCs recruited to the dental pulp**

It is known that dental pulp tissue receives various stimuli even under physiological conditions. As a result, degenerative changes, such as atrophy are induced in the dental pulp cells. Moreover, activation of odontoblasts occurs because of stimulation, and new dentin is formed [28]. Histopathological investigations have been performed for a long time on the changes in dental pulp cells caused by stimulation [29]. Since external mechanical stress is applied to the pulp tissue even during orthodontic tooth movement, tissue changes in the pulp have been investigated experimentally [30–32]. Some studies have been performed using electron microscopy [33]. However, previous studies of dental pulp cell differentiation have not considered the presence of BMDCs in the pulp. Recently, many findings have been clarified regarding the factors that control cell differentiation regulation, and factors related to the differentiation of hard tissue cells, such as osteoblasts, have also been investigated. Sigehara et al. reported the gene expression status of osteocalcin, osteopontin, and HSPs [34]. Nakano et al. [35] investigated cell differentiation in the pulp caused by orthodontic mechanical stress using immunohistochemical techniques. The results of the study showed that the expression of Runx2 increased in the odontoblasts in the pulp, and the activity of odontoblasts may be increased. Runx2 is a transcriptional regulator belonging to the Runx family, including Runx1, Runx2, and Runx3 [30]. The Runx family is involved in cell differentiation and cell cycle progression [31] and is an essential transcriptional regulator of osteoblast differentiation and development [32]. Runx2 is a master regulator of odontoblast differentiation as well as osteoblasts [28]. No bone tissue was produced in the pulp. Therefore, the expression of Runx2 in the dental pulp strongly suggests odontoblast differentiation. Additionally, ALP expressed in the early stage of calcification associated with odontoblast differentiation is an index of odonoblastic activity [33]. In our previous studies, we induced the differentiation of dental pulp cells by adding injurious stimulation to the periodontium and pulp [10, 11]. Nabeyama et al. have shown that there is a weak positive reaction for Runx2 in the pulp tissue that has not been artificially stimulated [10]. This is similar to the results of the study conducted by Nakano et al. [35, 36], and it is presumed that it is a reaction to physiological stimuli, such as mastication and occlusion. Furthermore, Nabeyama et al. observed tissue changes in the pulp by stimulating the pulp with the immediate teeth separation method used for dental treatment for 30 min and observed the expression of Runx2 in odontoblasts, vascular endothelial cells of the pulp, and pulp-specific cells [10]. Additionally, its expression reached the maximum levels after 24 h, then gradually disappeared, and decreased to the same level as that of the control group at 1 week. It is considered that this is because BMDCs are recruited to the pulp and differentiated into pulp cells slightly after the local stimulation.

Regarding tooth separation in conservative dental treatment, the mechanical stress generated by the operation puts a stress load on the periodontal tissue,

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

especially on the periodontal ligament [37]. However, at the same time, it is easy to understand that it also acts as an injury stimulus on the pulp tissue of the tooth. That is, compression of the periodontal ligament in the alveolar region causes strong injurious stimulation of the vessels and nerves. Because these vessels and nerves are connected to the pulp, the damaging stimulus propagates into the pulp. However, there has been little research on the type of damage that actually occurs in dental pulp cells, and within the scope of our literature, we mention the expression of HSPs in dental pulp tissue. Only a few papers have been published regarding this issue. Sens et al. immunohistochemically examined the expression of HSPs in the pulp of human third molars and examined odontoblasts, dentin cell projections, dental fibroblasts, pulp vascular endothelial cells, and some blood vessels. HSPs are expressed in membranous smooth muscle cells [38]. Additionally, Tate et al. and Suzuki et al. experimentally verified the expression of HSPs in odontoblasts of the pulp after laser cavity formation in dental conservative treatment [39, 40]. Furthermore, Matsuzaki et al. reported a study at the mRNA level that HSP27 increased in the pulp with aging [41]. There are also forecast papers that investigated the expression of HSP27 and Runx2 in the pulp under orthodontic mechanical stress loading [35, 36]. The results of these studies indicated that HSP27 expression is associated with the differentiation of dental pulp cells into odontoblasts. It is widely known that HSPs are expressed in response to injurious stimuli, and they work to maintain homeostasis of the injured tissue. HSPs are proteins acquired for survival in a harsh environment in which cells are placed. They are strongly induced by non-physiological stimuli and have anti-apoptotic functions as molecular chaperones [42]. In addition, it is known that most HSPs are expressed as a cell response to stress, suppress protein denaturation, and repair denatured proteins. In fact, HSPs are constitutively expressed even under nonstress conditions and are essential proteins for various cell activities, such as cell differentiation, proliferation, survival, and function maintenance, *in vitro* and *in vivo* [42]. HSPs, such as HSP70 and HSP90, are known to act as molecular chaperones that temporarily bind to immature proteins, mediate the folding and association of polypeptides, and assist in protein maturation [43]. It is speculated that low molecular weight HSPs also function as molecular chaperones in cells, but the details have not been clarified [44]. HSP27 was initially discovered as an inhibitor of actin polymerization [45]. Since then, it is known that HSP27 is present in high concentrations in cells, such as skeletal muscle cells and vascular smooth muscle cells, even in the non-stimulated state. Thus, it is considered that HSP27 plays a physiological role in the vascular system [46].

Saito et al. stimulated the pulp by the same experimental method as Nabeyama et al. and examined the expression of HSP27 [11]. The pulp showed a weak positive reaction for HSP27 from the immunohistochemical reaction of the control group. These results are similar to the experimental results by Nakano et al. [35], and it is inferred that this may be a reaction to the constant load of physiological mechanical stress, such as mastication and tongue pressure on the teeth. The HSP27-positive reaction observed in the pulp of the control group was weakly expressed in the pulp cells but was mainly expressed in the vascular endothelial cells. This indicates that HSP27 plays a physiological role in the vascular system [46]. Next, HSP27 was strongly expressed in the vascular endothelial cells of the dental pulp 30 min after the teeth separation treatment and was also observed in some odontoblasts. Additionally, this expression reached its maximum after 24 h, then gradually disappeared, and its expression decreased to the same level as that of the control group at 1 week. It is considered that this is because dental pulp cells are stimulated by the stress of teeth separation, and HSP27 is expressed as a vascular reaction to it and induction of odontoblast differentiation.

Since all cells derived from GFP transgenic mice express GFP, all bone marrow cells of GFP bone marrow transplanted mice also express GFP. BMDCs are recruited to various places and transformed into various cells. However, we can track BMDCs using GFP as a marker [26, 27]. We used an experimental system using GFP mice to perforate the pulp cavity of the maxillary left first molar. The growth of granulation tissue in the pulp was examined to clarify the origin of the cellular components involved in the formation of periodontal polyps. In other words, a large number of BMDCs were recruited to the pulp that has been stimulated by inflammation. BMDCs are thought to differentiate into various cells in the pulp. BMDCs appear not only in the pulp but also in the periodontal ligament. Cells with spindle-shape cells, blood vessels, and polynuclear giant cells that make up the periodontal ligament were positive for GFP. There were no GFP-positive cells among the epithelial cells, but it was found that the GFPpositive cells infiltrated the epithelial tissue. Therefore, GFP-positive cells are considered dendritic cells.

The cells in the capillaries of the periodontal polyp prepared by Osuka's experimental model can be identified by immunohistochemical examination of CD31 as a marker of vascular endothelial cells. The highest number of capillaries was observed at 3 months. Immunofluorescence double staining of GFP and S100A4 in tissues over 2 weeks to 6 months detected green fluorescence (**Figure 5-a**) showing GFP-positive cells and red fluorescence showing S100A4 positive fibroblast-like spindle-shaped cells. (**Figure 5-b**). The region with orange fluorescence included cells with both proteins (**Figure 5-c**). As a result of marking the nucleus with DAPI, orange fluorescence was observed around the nucleus that usually emits blue fluorescence (**Figure 5d**).

Immunofluorescent double staining for GFP and Runx2 detected both oval and spindle-shaped cells positive for GFP green fluorescence (**Figure 6-a**) and Runx2

#### **Figure 5.**

*FIHC images of periodontal polyp (a: GFP; b: S100A4; c: Merged image of GFP and S100A4; and d: Merged image of S100A4, GFP and DAPI; 2 week specimen; scale bar = 20* μ*m).*

**Figure 6.**

*FIHC images of periodontal polyp (a: GFP; b: Runx2; c: Merged image of GFP and Runx2; and d: Merged image of Runx2, GFP and DAPI; 2 week specimen; scale bar = 20* μ*m).*

red fluorescence (**Figure 6-b**). In the superimposed image, cells showing orange fluorescence coexpressed two proteins (**Figure 6-c**). When the image was overlaid with DAPI, which showed blue fluorescence in the nucleus, orange fluorescence was seen in the cytoplasm (**Figure 6-d**). Runx2-positive and GFP-positive cells that showed orange fluorescence were observed at 2 weeks to 1 month of periodontal polyp formation and then decreased at 3 months. Even at 6 months, both Runx2- and GFP-positive cells appeared. However, there were many cells that were individually stained with GFP. Normally, no bone tissue is formed in the pulp. Runx2-positive cells in the pulp mean odontoblasts. This result indicates that the cells that proliferate in the pulp and differentiate into Runx2-positive odontoblasts are GFP-positive BMDCs.
