**5. Notch signaling in cell differentiation of the BMDCs in the PDL**

The GFP mouse model examination revealed that the cells were derived from mesenchymal cells of the bone marrow. Furthermore, these cells differentiated into the tissue-specific PDL fibroblasts, blood capillary endothelial cells, etc. Notch is a membrane-bounded protein, which regulates the differentiation gen for changing the cell type [49]. However, there have been no reports on the component cells of periodontal polyp-granulation tissues [9].

In usual dental clinical practice, perforation of the floor of the dental pulp suddenly occurred during a dental treatment. In case of a large perforation, it cause chronic granulomatous growth in the regional portions [9]. Granulation tissue grows in the periodontal ligament (PDL) region from the perforated dentin causing periodontal polyp. Regarding the PDL polyp, our previous histopathological and immunohistochemical examinations were done [4–6]. The data using an experimental system on GFP mouse bone marrow transplantation model revealed that the cells were derived from mesenchymal cells of the bone marrow. Furthermore, these cells differentiated into the tissue-specific PDL fibroblasts, blood capillary endothelial cells, etc. (**Figure 6**).

In general, Notch signaling is necessary for cell fate determination, cell proliferation, and differentiation [56, 57]. Therefore in this study, we examined using the method of Matsuda, et al. [9]. We examine the expression of Notch in the experimentally induced pulp polyp component cells, using observation of histopathology and immunohistochemistry methods. Histopathological observation of 2-week specimens, the spindle-shaped cell proliferation was evident with some neutrophils in the specimens. Within these cells, the relatively round nucleus-having cell and some capillaries were observed.

The IHC examination of Notch expression of the 2-week specimens, elongated, and spindleshaped cells with spindle nucleus were positive to Notch1 (**Figure 7a, b**). From 1 to 6-month specimens, the spindle-shaped cells were also positive to Notch1. Notch1-positive reaction was continuously detected (**Figure 7c, d**). In contrast, as observed in the control, the dental pulpal tissues of the non-treated teeth were completely negative, although some nonspecific positive reactions existed (**Figure 7e, f**). Furthermore, the physiological PDL was slightly positive to Notch (**Figure 7e**).

extracellular form were not detected, but the number of cells increased in a short period of 1 week. To determine if an increase in cell number is made by migration of BMDCs, using

The number of GFP-positive cells immediately after stress load showed a gradual increase. It had increased over time until 6 months. It is certain that BMDCs were supplied to the PDF for a long period of time. Furthermore, when each cell was characterized by double immunofluorescence staining, Tsujigiwa et al. [1, 2] showed that the transplanted BMDCs, GFP-positive cells such as osteoclasts, were stained and identified. This confirmed that it moved and dif-

Applying the method of Muraoka [48], cell identification by double immunofluorescence staining with GFP-CD31, GFP- CD68, and GFP- Runx2 were performed. As a result, it was possible to distinguish between osteoclasts and macrophages. Furthermore, since some GFPpositive cells expressing CD31 were found, they were derived from BMDCs and differentiated into hemangioendothelial cells. Similar results were obtained for macrophages by CD68. Furthermore, since Runx2 represented fibroblasts [3, 4], respectively, the expression of Runx2 was executed. As a result, it was clear that it was GFP-positive and expression of Runx2, and the cell morphology was a certain swimming; so it was a PLD fibroblast which strongly sug-

**5. Notch signaling in cell differentiation of the BMDCs in the PDL**

been no reports on the component cells of periodontal polyp-granulation tissues [9].

The GFP mouse model examination revealed that the cells were derived from mesenchymal cells of the bone marrow. Furthermore, these cells differentiated into the tissue-specific PDL fibroblasts, blood capillary endothelial cells, etc. Notch is a membrane-bounded protein, which regulates the differentiation gen for changing the cell type [49]. However, there have

In usual dental clinical practice, perforation of the floor of the dental pulp suddenly occurred during a dental treatment. In case of a large perforation, it cause chronic granulomatous growth in the regional portions [9]. Granulation tissue grows in the periodontal ligament (PDL) region from the perforated dentin causing periodontal polyp. Regarding the PDL polyp, our previous histopathological and immunohistochemical examinations were done [4–6]. The data using an experimental system on GFP mouse bone marrow transplantation model revealed that the cells were derived from mesenchymal cells of the bone marrow. Furthermore, these cells differentiated into the tissue-specific PDL fibroblasts, blood capillary

In general, Notch signaling is necessary for cell fate determination, cell proliferation, and differentiation [56, 57]. Therefore in this study, we examined using the method of Matsuda, et al. [9]. We examine the expression of Notch in the experimentally induced pulp polyp component cells, using observation of histopathology and immunohistochemistry methods. Histopathological observation of 2-week specimens, the spindle-shaped cell proliferation was evident with some neutrophils in the specimens. Within these cells, the relatively round

Tomida's method [47], GFP-positive cells were counted.

ferentiated into the bone remodeling site.

104 Histology

gested that it had migrated from the bone marrow.

endothelial cells, etc. (**Figure 6**).

nucleus-having cell and some capillaries were observed.

Generally Notch is an important regulation signaling of morphogenesis. It was reported that Notch1 is a transmembrane protein necessary for cell fate determination, etc. [49]. Thus, we examined the relationship between the cell differentiation in the periodontal polyp component cells and Notch signaling in the present study. According to the present results: (1) spindleshaped fibroblastic cell of the pulp polyp tissues was almost Notch1-positive reactive and (2)

**Figure 7.** IHC images of notch expression. (a) Granulation tissue area, scale bar = 50 μm; (b) granulation tissue area, scale bar = 50 μm; (c) fibrous rich area, scale bar = 100 μm; (d) enlarged view of c, scale bar = 50 μm; (e) control areas (dental pulp and periodontal ligament tissues), scale bar = 100 μm; and (f) control areas (dental pulp and periodontal ligament tissues), scale bar = 50 μm. Quotation alteration of #44.

these reactions strongly suggested that the cell differentiation was caused by the Notch1 signaling. The reactions mean that the PDL is always received and controlled by Notch signaling.

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