**3. GFP-BMDCs into the PDL received occlusal trauma**

Regarding the examination results of periodontal ligament in experimental occlusal trauma mouse model, we have reported the cytological behavior of the related regions. The experimental model diagram is shown in **Figure 3**. Periodontal connective tissue remodeling occurred due to traumatic occlusal overload [31]. In the remodeling course, the fibroblasts act as an important role. In the process of PDL remodeling phenomenon, fibroblasts make an important role such as collagen synthesis. Heat shock protein 47 (HSP 47) is a protein that acts as a molecular chaperone in procollagen biosynthesis and maturation. Type I collagen is a major component of PDL. Therefore, in our study, the expression of IHC of HSP 47 on the experimental-induced periodontal tissue of traumatic occlusion was investigated. That is, an experimental occlusal trauma model was developed and experimented. Expression of HSP in occlusal trauma periodontal tissue was performed using immunohistochemistry (IHC). The results indicated that fibroblasts had high HSP expression in response to excessive traumatic occlusion. HSP 47 is thought to play an important role in the maintenance of fibroblast homeostasis exposed to traumatic occlusion. HSP 27 and HSP 70 have detailed observation results on damaged periodontal tissue by Muraoka et al. [5–7]. Based on the experiments of Fujii et al. [31] and Takaya et al. [32], we carried out the IHC study on the histopathological changes of mouse dental tissue and on HSP.

The experimental outline is shown in **Figure 3**. After that, changes in the periodontal ligament were observed over time. Then after, the micro plus screw was removed at day 4 after implantation, and the subsequent tissue changes were observed. Histopathological examination: It was observed that a fibroblast and a spindle-shaped cell have high density in a periodontal ligament of the control group. An erythrocyte was filled by a capillary. Periodontal ligament

Bone Marrow Mesenchymal Cell Contribution in Maintenance of Periodontal Ligament… http://dx.doi.org/10.5772/intechopen.80785 99

**Figure 3.** Diagram of the mouse traumatic model.

GFP-positive cells were detected in PDL in both experimental and control groups in this study. In the experimental group, a number of GFP-positive cells were found in the PDL tissue and intermittently stimulated intermittent mechanical stress. However, there were few GFP-positive cells in the control group. This result was significantly larger between the experimental group and the control group. This suggests that orthodontic mechanical stress

BMC migrate from bone marrow tissue and different types of tooth-related cell types including odontoblasts [27], osteoclasts [28], and PDL [29, 30]. Osteoblasts and osteoclasts maintain and reconstruct cancellous bone surrounding the marrow tissue. BMCs from the bone marrow are closely involved in the repair of tissues to maintain periodontal tissue homeostasis of PDL fibroblasts. Furthermore, mechanical stress strongly induces cellular activation of these PDLs. Teeth can be produced from non-odontogenic stem cells. This establishes the basic principle that bone marrow stem cells are also involved in tooth embryogenesis. As a future therapeutic possibility, these cells include transplantation to a tooth defect site or transplantation into a patient's bone marrow with developmental abnormality, which may lead to a new

Regarding the examination results of periodontal ligament in experimental occlusal trauma mouse model, we have reported the cytological behavior of the related regions. The experimental model diagram is shown in **Figure 3**. Periodontal connective tissue remodeling occurred due to traumatic occlusal overload [31]. In the remodeling course, the fibroblasts act as an important role. In the process of PDL remodeling phenomenon, fibroblasts make an important role such as collagen synthesis. Heat shock protein 47 (HSP 47) is a protein that acts as a molecular chaperone in procollagen biosynthesis and maturation. Type I collagen is a major component of PDL. Therefore, in our study, the expression of IHC of HSP 47 on the experimental-induced periodontal tissue of traumatic occlusion was investigated. That is, an experimental occlusal trauma model was developed and experimented. Expression of HSP in occlusal trauma periodontal tissue was performed using immunohistochemistry (IHC). The results indicated that fibroblasts had high HSP expression in response to excessive traumatic occlusion. HSP 47 is thought to play an important role in the maintenance of fibroblast homeostasis exposed to traumatic occlusion. HSP 27 and HSP 70 have detailed observation results on damaged periodontal tissue by Muraoka et al. [5–7]. Based on the experiments of Fujii et al. [31] and Takaya et al. [32], we carried out the IHC study on the histopathological

The experimental outline is shown in **Figure 3**. After that, changes in the periodontal ligament were observed over time. Then after, the micro plus screw was removed at day 4 after implantation, and the subsequent tissue changes were observed. Histopathological examination: It was observed that a fibroblast and a spindle-shaped cell have high density in a periodontal ligament of the control group. An erythrocyte was filled by a capillary. Periodontal ligament

induces GFP-positive transplanted BMCs into the PDL tissues.

**3. GFP-BMDCs into the PDL received occlusal trauma**

approach to tooth and jaw bone regeneration.

98 Histology

changes of mouse dental tissue and on HSP.

fiber was arranged irregularly. An osteoclast could see conspicuously in an alveolar bone. The furcation was lined by acellular cementum. At experimental group on day 1, the capillary which swelled was filled with an erythrocyte. The amount of deeply stained cells with round nuclei increased. More osteoclasts were observed on the glassy surface of the alveolar bone.

There are several kinds in a stem cell; a stem cell differentiates into various cells of a human body and has a special nature. The stem cell and a marrow-derived cells (BMCs) also possess the differentiation special quality of the plural. Many researchers reported that BMCs might relate into retinal vessels, myoblasts, hepatocytes in the liver, Purkinje neurons, cardiac muscle in the heart, and airway epithelial cells in recent years [33, 34]. A stem cell can be used in the field of the regenerative medicine; so to regenerate an organ, the stem cell is very important for treatment of various diseases [35]. For treatment of an end limb ischemia and an ischemia disease including myocardial infarction, a try at a local delivery of BMCs is studied [36, 37].

It is stated that occlusal trauma is defined as damage resulting from tissue changes within the PDL as a result of abnormal occlusion forces. It has been proven for many years by many researchers that occlusal trauma may cause various destructive biological reactions to the tissue of PDL [37–40]. A lot of researchers reported cytological kinetic examinations of PDL tissue regarding occlusal trauma PDL, but they have not been fully performed regarding establishing an experimental system with animals that can be used in a very versatile manner; we have constructed an experimental system with a mouse with respect to the occlusal trauma model. We reviewed the organization of PDL from the perspective of cytological kinetics [40]. We then performed a histopathological and also immunohistochemical study. **Figure 4** shows histopathology and IHC results.

control groups in the histopathological photographs. The result was markedly increased on the day 4 of the experimental group. Compared with the experimental group, experimental group on day 7 and 14 decreased but mostly of the same degree share, and they were not

Bone Marrow Mesenchymal Cell Contribution in Maintenance of Periodontal Ligament…

http://dx.doi.org/10.5772/intechopen.80785

101

GFP-positive cells were sparse in the control group and the experimental group on days 4 and 14. These cellular contours are PDL cells with circular nuclei. According to the digital image analysis method, the number of GFP-positive cells increased in the experimental group day 7. The results of image analysis of GFP-positive cells of PDL on the day 7 of the experimental group

In the progress of periodontal disease [41], things such as dental plaque and tartar caused by tooth deposits are common, but it is well-known that occlusion abnormalities such as traumatic occlusion are also important. Histopathological examination of PDL has been conducted [42–46] so far. These were done using rats, mice, macaque monkeys, and Beagle dogs. However, the report did not find a focus point at cytological kinetics of periodontal ligament due to excessive occlusal loading. Thus, we focused the cytological kinetics in the periodontal

GFP-IHC specimens shows, although the positive rate of GFP was considerably high on the day 7 of the experimental group, that there was almost no significant difference in the day 14 of the experimental group as compared with the control group. All cells constituting individual tissue cells of GFP transgenic mice have green fluorescent protein. Even if any types of cell differentiate transplanted bone marrow-derived cells, they can be traced by GFP. It is a technique of immunofluorescent staining. Such cells were identified from bone marrow-transplanted cells. At first it was osteoclasts and macrophages. It is reported later in the results of the previous experiment that many GFP-positive cells migrate to the PDL tissues in mice and differentiate into in situ specific cells. Furthermore, it is easy to imagine that dendritic cells and PDL fibroblasts, which migrate into the PDL, differentiate into PDL-

In the experiment, bone marrow-derived cells showing GFP-positive cells in PDL of the root bifurcation region subjected to occlusal trauma on the day 7 of experimental group increased. It is clear that the majority of GFP-positive cells are osteoclasts and macrophages by previous studies. It is not only PDL damage due to continuous excessive occlusal trauma. It causes remodeling of alveolar bone and PDL and mobilization of bone marrow-derived cells is neces-

According to many studies, PDL remodeling phenomenon due to acceleration of cell activation is caused by excessive traumatic occlusion stress on the day 4 of the experimental group on the tooth PDL damaged part in the root furcation region. In the experimental group day 7, PDL remodeling mechanism is done by osteoclasts and macrophages. The cells are present in the furcation area as GFP-positive BMDCs. Therefore, when gingivitis has not been caused at all or only very slightly, the PDL stressed with traumatic occlusion; it will be

showed a considerably larger increase in comparison with the control group.

significant to compare with the control group.

tissues by excessive occlusal loading.

specific cells.

sary for it.

constructed by BMDCs.

**Figure 4.** Histopathology of control region (a) and experimental region (b) of the day 4 specimen. Scale bar = 50 μm. IHC of GFP. Control specimen (c) and experimental day 7 specimen (d). Scale bar = 50 μm. Quotation alteration of literature #32.

Eleven 7-week-old ddY male mice and eight 7-week-old bone marrow-transplanted female C57BL/6 genealogy mice from GFP transgenic mice (GFP mice), for a total of 19 mice, were used in this study. Histopathological examination showed as followed. Control group specimens showed the PDL maintained a constant width; the major fibers arranged in orderly cementum and alveolar bone. Spindle-shaped fibroblasts that appeared in PDL were collagen fiber bundles. In the relatively dense cell nucleus, PDL, there was a congested capillary. Furthermore, the cellular cementum could be clearly confirmed. On day 4 of the experimental group, PDL was somewhat compressed, and evident capillary vessel filling was confirmed. In spindle-shaped fibroblasts, those with deeply stained circular nuclei of hematoxylin increased in number. Multinucleate giant cells appeared mainly on the alveolar bone surface. It gradually absorbed bone tissue and made some blanks. In the experimental group, the circular nucleus cells decreased considerably on day 7 from day 4. Vascular hypertrophy developed. Multinucleate giant cells were expressed on the alveolar bone surface of Howship's lacunae. On day 4 of the experimental group specimens, the regression of hyaline degeneration area had expanded. Furthermore, the cellular cementum destruction was evident in the expansion of PDL areas. On day 14 of the experimental group, the cementum absorption region by multinucleated giant cells and the alveolar bone surface rapidly expanded remarkably. There was a decrease in cells with circular nuclei. Cells in which both the nucleus and the cytoplasm are spindle-shaped are increasing again. The width of PDL became wider.

Using the cytological kinetics method, we analyzed the nuclear occupancy to compare all cell numbers. The area examining the occupancy rate analyzed related PDL experiments and control groups in the histopathological photographs. The result was markedly increased on the day 4 of the experimental group. Compared with the experimental group, experimental group on day 7 and 14 decreased but mostly of the same degree share, and they were not significant to compare with the control group.

GFP-positive cells were sparse in the control group and the experimental group on days 4 and 14. These cellular contours are PDL cells with circular nuclei. According to the digital image analysis method, the number of GFP-positive cells increased in the experimental group day 7. The results of image analysis of GFP-positive cells of PDL on the day 7 of the experimental group showed a considerably larger increase in comparison with the control group.

In the progress of periodontal disease [41], things such as dental plaque and tartar caused by tooth deposits are common, but it is well-known that occlusion abnormalities such as traumatic occlusion are also important. Histopathological examination of PDL has been conducted [42–46] so far. These were done using rats, mice, macaque monkeys, and Beagle dogs. However, the report did not find a focus point at cytological kinetics of periodontal ligament due to excessive occlusal loading. Thus, we focused the cytological kinetics in the periodontal tissues by excessive occlusal loading.

GFP-IHC specimens shows, although the positive rate of GFP was considerably high on the day 7 of the experimental group, that there was almost no significant difference in the day 14 of the experimental group as compared with the control group. All cells constituting individual tissue cells of GFP transgenic mice have green fluorescent protein. Even if any types of cell differentiate transplanted bone marrow-derived cells, they can be traced by GFP. It is a technique of immunofluorescent staining. Such cells were identified from bone marrow-transplanted cells. At first it was osteoclasts and macrophages. It is reported later in the results of the previous experiment that many GFP-positive cells migrate to the PDL tissues in mice and differentiate into in situ specific cells. Furthermore, it is easy to imagine that dendritic cells and PDL fibroblasts, which migrate into the PDL, differentiate into PDLspecific cells.

Eleven 7-week-old ddY male mice and eight 7-week-old bone marrow-transplanted female C57BL/6 genealogy mice from GFP transgenic mice (GFP mice), for a total of 19 mice, were used in this study. Histopathological examination showed as followed. Control group specimens showed the PDL maintained a constant width; the major fibers arranged in orderly cementum and alveolar bone. Spindle-shaped fibroblasts that appeared in PDL were collagen fiber bundles. In the relatively dense cell nucleus, PDL, there was a congested capillary. Furthermore, the cellular cementum could be clearly confirmed. On day 4 of the experimental group, PDL was somewhat compressed, and evident capillary vessel filling was confirmed. In spindle-shaped fibroblasts, those with deeply stained circular nuclei of hematoxylin increased in number. Multinucleate giant cells appeared mainly on the alveolar bone surface. It gradually absorbed bone tissue and made some blanks. In the experimental group, the circular nucleus cells decreased considerably on day 7 from day 4. Vascular hypertrophy developed. Multinucleate giant cells were expressed on the alveolar bone surface of Howship's lacunae. On day 4 of the experimental group specimens, the regression of hyaline degeneration area had expanded. Furthermore, the cellular cementum destruction was evident in the expansion of PDL areas. On day 14 of the experimental group, the cementum absorption region by multinucleated giant cells and the alveolar bone surface rapidly expanded remarkably. There was a decrease in cells with circular nuclei. Cells in which both the nucleus and the cytoplasm

**Figure 4.** Histopathology of control region (a) and experimental region (b) of the day 4 specimen. Scale bar = 50 μm. IHC of GFP. Control specimen (c) and experimental day 7 specimen (d). Scale bar = 50 μm. Quotation alteration of literature #32.

100 Histology

are spindle-shaped are increasing again. The width of PDL became wider.

Using the cytological kinetics method, we analyzed the nuclear occupancy to compare all cell numbers. The area examining the occupancy rate analyzed related PDL experiments and In the experiment, bone marrow-derived cells showing GFP-positive cells in PDL of the root bifurcation region subjected to occlusal trauma on the day 7 of experimental group increased. It is clear that the majority of GFP-positive cells are osteoclasts and macrophages by previous studies. It is not only PDL damage due to continuous excessive occlusal trauma. It causes remodeling of alveolar bone and PDL and mobilization of bone marrow-derived cells is necessary for it.

According to many studies, PDL remodeling phenomenon due to acceleration of cell activation is caused by excessive traumatic occlusion stress on the day 4 of the experimental group on the tooth PDL damaged part in the root furcation region. In the experimental group day 7, PDL remodeling mechanism is done by osteoclasts and macrophages. The cells are present in the furcation area as GFP-positive BMDCs. Therefore, when gingivitis has not been caused at all or only very slightly, the PDL stressed with traumatic occlusion; it will be constructed by BMDCs.
