**4. Differentiation of BMDCs into the PDL peculiar cells**

The IHC view is as follows. BMDC is GFP-positive except for an endothelial cell of microcapillarity. PDL cells and/or cells spindle in shape on the alveolar bone surface are mostly GFP-positive (**Figure 5a**). Furthermore, the micro-capillaries are positive for GFP in some occasions of the periphery (**Figure 5b**).

Cells of various cell types could be identified by immunofluorescence double staining check for GFP. Green fluorescent GFP-positive cells as orange fluorescence by fluorescence immunohistochemical double staining of GFP-S100A4 (**Figure 5c**) and GFP-Runx2 were consistent with red fluorescent S-100 A 4-positive and Runx2-positive cells (**Figure 5d**). Fusiform cells toward the root direction were arranged in a bundle. When superimposing, the nucleus stained blue, and the cell of that cell was stained orange. The enlarged view of **Figure 6** also clearly shows both positive for GFP and CD 31 and proposes PDL capillaries (**Figure 5e**).

Recently, many studies using undifferentiated mesenchymal cells (UDMC) of the bone marrow are done in the repair and regeneration of tissues, bones, and other organs around the

teeth. Regarding the alveolar bone and the PDL regenerations, it was described for the differentiation of UDMC from bone marrow into cartilage [11, 12]. Experiments on BMDCs using GFP mice showed that after cell transplantation, it differentiated into salivary gland epithelial cells and myoepithelial cells capable of playing a role not only of olfactory cells but also salivary gland tissue [13]. Histopathological examination of experimentally loaded mice in orthodontic treatment showed that bone marrow stem cells increase bone remodeling capacity [3].

**Figure 6.** Micro-CT of day 4 specimen (a), histopathology view (b, c), and IHC of GFP (d). Micro-CT of day 7 specimen

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(e), histopathology view (f, g), and IHC of GFP (h). Scale bar = 50 μm. Quotation alteration of #44.

Tomida et al. reported that PDL cells increased after 1–5 weeks of mechanical stress. In the current study, the cell number was calculated by counting GFP-positive cells in the control group and the experimental group [47]. The results showed that the number of GFP-positive cells in the experimental group was much higher than that in the control group. The increase in the number of cells in PDL after mechanical stress loading strongly suggests that it occurred because BMDCs were transferred to PDL. Using GFP mouse experiments, Tsujigiwa and others indicated that the transplanted BMDCs migrated to pulp and differentiated into cells constituting pulp tissue [1]. By using the same experiment, Muraoka et al. [48] showed that the transplanted BMDCs migrated to PDLs and later differentiated into PDL cells. However, the biological response of mesenchymal cells in response to mechanical stimuli also caused changes in cell morphology [48]. Movement of transplanted BMDCs using GFP mice was

The results of the study showed that the mechanical stress promoted the increase in the number of cells in both pressure and tension sides of the PDL. Morphological changes of the

reported to be due to mechanical stress [49].

**Figure 5.** IHC results showing GFP-positive round- or spindle-shaped cells within the PDL tissues (a, scale bar = 50 μm) and GFP-positive products existing within the vascular endothelial cells (b, scale bar = 50 μm). Regarding the lower layer photographs (c–e), enlarged FIHC images of PDL, 6 months specimen. GFP (green) and merged image (c) of S-100 A4 (red) result fibroblast. GFP (green) and Runx2 (red) view (d) shows clearly both positive suggesting PDL fibroblasts, and GFP (green) and CD31 (red) image (e) suggests endothelial cells. Scale bar = 100 μm. Quotation alteration of literatures #26 and 43.

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**4. Differentiation of BMDCs into the PDL peculiar cells**

occasions of the periphery (**Figure 5b**).

102 Histology

#26 and 43.

The IHC view is as follows. BMDC is GFP-positive except for an endothelial cell of microcapillarity. PDL cells and/or cells spindle in shape on the alveolar bone surface are mostly GFP-positive (**Figure 5a**). Furthermore, the micro-capillaries are positive for GFP in some

Cells of various cell types could be identified by immunofluorescence double staining check for GFP. Green fluorescent GFP-positive cells as orange fluorescence by fluorescence immunohistochemical double staining of GFP-S100A4 (**Figure 5c**) and GFP-Runx2 were consistent with red fluorescent S-100 A 4-positive and Runx2-positive cells (**Figure 5d**). Fusiform cells toward the root direction were arranged in a bundle. When superimposing, the nucleus stained blue, and the cell of that cell was stained orange. The enlarged view of **Figure 6** also clearly shows both positive for GFP and CD 31 and proposes PDL capillaries (**Figure 5e**).

Recently, many studies using undifferentiated mesenchymal cells (UDMC) of the bone marrow are done in the repair and regeneration of tissues, bones, and other organs around the

**Figure 5.** IHC results showing GFP-positive round- or spindle-shaped cells within the PDL tissues (a, scale bar = 50 μm) and GFP-positive products existing within the vascular endothelial cells (b, scale bar = 50 μm). Regarding the lower layer photographs (c–e), enlarged FIHC images of PDL, 6 months specimen. GFP (green) and merged image (c) of S-100 A4 (red) result fibroblast. GFP (green) and Runx2 (red) view (d) shows clearly both positive suggesting PDL fibroblasts, and GFP (green) and CD31 (red) image (e) suggests endothelial cells. Scale bar = 100 μm. Quotation alteration of literatures

**Figure 6.** Micro-CT of day 4 specimen (a), histopathology view (b, c), and IHC of GFP (d). Micro-CT of day 7 specimen (e), histopathology view (f, g), and IHC of GFP (h). Scale bar = 50 μm. Quotation alteration of #44.

teeth. Regarding the alveolar bone and the PDL regenerations, it was described for the differentiation of UDMC from bone marrow into cartilage [11, 12]. Experiments on BMDCs using GFP mice showed that after cell transplantation, it differentiated into salivary gland epithelial cells and myoepithelial cells capable of playing a role not only of olfactory cells but also salivary gland tissue [13]. Histopathological examination of experimentally loaded mice in orthodontic treatment showed that bone marrow stem cells increase bone remodeling capacity [3].

Tomida et al. reported that PDL cells increased after 1–5 weeks of mechanical stress. In the current study, the cell number was calculated by counting GFP-positive cells in the control group and the experimental group [47]. The results showed that the number of GFP-positive cells in the experimental group was much higher than that in the control group. The increase in the number of cells in PDL after mechanical stress loading strongly suggests that it occurred because BMDCs were transferred to PDL. Using GFP mouse experiments, Tsujigiwa and others indicated that the transplanted BMDCs migrated to pulp and differentiated into cells constituting pulp tissue [1]. By using the same experiment, Muraoka et al. [48] showed that the transplanted BMDCs migrated to PDLs and later differentiated into PDL cells. However, the biological response of mesenchymal cells in response to mechanical stimuli also caused changes in cell morphology [48]. Movement of transplanted BMDCs using GFP mice was reported to be due to mechanical stress [49].

The results of the study showed that the mechanical stress promoted the increase in the number of cells in both pressure and tension sides of the PDL. Morphological changes of the 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 Tomida's method [47], GFP-positive cells were counted.

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

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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.

positive to Notch (**Figure 7e**).

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 differentiated into the bone remodeling site.

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 suggested that it had migrated from the bone marrow.
