**2. GFP-BMDCs into the PDL-received orthodontic mechanical stress**

The periodontal ligament (PDL) is usually remodeling at physiological in condition. Furthermore, orthodontic treatment results to mechanical stress inducing reorganization of PDL collagen bundles. The examination results in "movement of an orthodontics tooth." The mechanical stress communicated to PDL causes a reaction of organization and causes "movement of a tooth." This reaction of PDL is the one for maintenance of a homeostasis. A histology-like reply and production of the copying factor which controls a cell differentiation and various morphogenesis phenomena are studied more widely in recent years [3, 4]. It becomes clear that it's able to bring manifestation of a remodeling of a periodontal tissue and the activated molecule which replies to various mechanical stress and inflammation to maintain a homeostasis [5–10]. Our experimental method was based on our previous reports [4, 5]. The Waldo method of inducing mechanical stress load in mouse periodontal tissues was followed. Under general anesthesia, the mouse was inserted between the maxillary molars to induce persistent mechanical stress. A separator was inserted between M1 and M2 of the right maxillary molars to ensure the mechanical stress due to pressure over a period of time. After each experimental time, the periodontal tissues of the left maxillary molar region (untreated side) were used as controls. In this experiment, the distal buccal root of the maxillary first molar was the observation part. The schematic diagram, macro-view, and histology are shown in **Figure 2**.

Therefore, we focused on the expression of various HSPs that maintain homeostasis during injury. HSPs are one of the factors recognized that is transiently enhanced by heat shock [11]. It is also called stress protein because it is not only enhanced by heat shock but also by ischemia; other pathological changes such as infections and inflammation and radiation; physical stress such as light; stress from enzymes, heavy metal ion, arsenic, arsenic acid, methanol, and active oxygen; and stress from chemical and various amino acid derivatives [12, 13]. When the chromosomes of the salivary gland of *Drosophila* were at high temperatures, HSP

express many cells in response to stress, suppression, and repair of proteins whose properties have been altered. In addition, HSPs are essential proteins to maintain various cellular life functions between cell differentiation and growth and presence. It is a widely distributed intracellular equilibrium protein, which is regularly expressed under various conditions in in vitro and in vivo experiments. Depending on the molecular weight, HSP is classified into high molecular HSP (HSP 110, HSP 90, HSP 70, and HSP 40–60 families) and low molecular HSP (HSP 20 family). These are polypeptides of tens to hundreds of kDa. Many researches have been done on expression and function of these HSPs and various sites. That is, high molecular HSPs such as HSP 70 and HSP 90 have a role of assisting the maturation of proteins. That is, it temporarily binds to immature state protein and acts as a molecular chaperone. However, small molecular HSPs that function as molecular chaperones have not yet been reported [21]. HSP 27 belongs to the low molecular HSP family. Firstly, HSP 27 was found in the actin polymerization. HSP 27 is known to exist at high levels in non-stimulated vascular smooth muscle and skeletal muscle cells. From this, it is believed that HSP 27 plays a role in maintaining blood pressure and other physiological effects in the vascular system [18–20].

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We reported that BMCs migrated into the PDL regions; PDL fibroblasts, hemendothelial cells,osteoclasts, and Langerhans cells migrated into the PDF regions. These cells differentiate into their tissue-specific cells [21–23]. The facts are that BMCs are not mesodermal cells and they are derived from neural crest cells. This differentiation into specialized cells may be done on the spot by expression of odontogenic genes [24]. According to a classical tissue engineering technique, the tooth-like structure is created based on biodegradable polymer scaffolds by transplantation of dental pulp cell pellets or BMC extracted directly from dental embryonic

We examined the transplanted BMC migration into the PDL. The IHC revealed that GFPpositive cells were detected in the PDL tissues. A number of GFP-positive cells appeared on mechanically loaded periodontal tissue, especially on the tension side of the experimental group. On the other hand, little GFP-positive cells appeared in the control group. From the above results, we analyzed how they differ from the experimental group and control group [25]. Thus, these data indicated that orthodontic mechanical stress acts as a possible promoting factor of transplanted bone marrow-derived cell migration into periodontal tissues and of

On the other hand, mice transplanted with bone marrow cells of GFP transgenic mice were used to observe GFP-positive cells in dental pulp of mouse incisors, PLD, oral epithelial Langerhans cells, pulp fibroblasts, dental vascular endothelial cells, and osteoclasts. GFPpositive cells in the dental pulp are dendritic cell-like cells, and some odontoblast-like cells also showed a positive response to GFP. It is clear that BMCs have the ability to differentiate into teeth and related connective tissues. GFP-positive cells histopathologically differentiated into several cell types. Fluorescent immunohistochemistry (FIHC) and tartrate-resistant acid phosphatase (TRAP) staining techniques showed that these cells were detected as osteoclasts and macrophages. In addition, GFP-positive cells gathered in adjacent blood vessels. This data suggests that GFP-positive BMCs migrate to periodontal tissues and differentiate PDL

cells and dental pulp stem cells separated by enzyme treatment [25].

differentiation to fibroblasts [26].

tissue-specified cells.

**Figure 2.** Experimental schema (a), macro-view (b, c), and histology (d). Quotation alteration of literature #5.

strongly expressed [14]. After that, the isolation of synthesized HSP by a SDS-polyacrylamide gel electrophoresis first by *Drosophila*. Some of the main protein which was also led by heat shock method of treatment in non-salivary glandular system was regarded as HSP together. A puff of the mRNA history of HSP was copied. After that a high study is reporting that the colon bacillus kept during the environment, leaven, and a gene of other mammalian cells are able to receive a heat stress and preserve a gene more than they are concerned with HSP [14]. The representation of these HSPs is then a common phenomenon [15]. HSPs have molecular ancillary and functional antiapoptotic capacity [16, 17] from ancient times. That is, it is a protein that develops to escape cell death in harsh conditions for cell survival. Many HSPs also express many cells in response to stress, suppression, and repair of proteins whose properties have been altered. In addition, HSPs are essential proteins to maintain various cellular life functions between cell differentiation and growth and presence. It is a widely distributed intracellular equilibrium protein, which is regularly expressed under various conditions in in vitro and in vivo experiments. Depending on the molecular weight, HSP is classified into high molecular HSP (HSP 110, HSP 90, HSP 70, and HSP 40–60 families) and low molecular HSP (HSP 20 family). These are polypeptides of tens to hundreds of kDa. Many researches have been done on expression and function of these HSPs and various sites. That is, high molecular HSPs such as HSP 70 and HSP 90 have a role of assisting the maturation of proteins. That is, it temporarily binds to immature state protein and acts as a molecular chaperone. However, small molecular HSPs that function as molecular chaperones have not yet been reported [21]. HSP 27 belongs to the low molecular HSP family. Firstly, HSP 27 was found in the actin polymerization. HSP 27 is known to exist at high levels in non-stimulated vascular smooth muscle and skeletal muscle cells. From this, it is believed that HSP 27 plays a role in maintaining blood pressure and other physiological effects in the vascular system [18–20].

We reported that BMCs migrated into the PDL regions; PDL fibroblasts, hemendothelial cells,osteoclasts, and Langerhans cells migrated into the PDF regions. These cells differentiate into their tissue-specific cells [21–23]. The facts are that BMCs are not mesodermal cells and they are derived from neural crest cells. This differentiation into specialized cells may be done on the spot by expression of odontogenic genes [24]. According to a classical tissue engineering technique, the tooth-like structure is created based on biodegradable polymer scaffolds by transplantation of dental pulp cell pellets or BMC extracted directly from dental embryonic cells and dental pulp stem cells separated by enzyme treatment [25].

We examined the transplanted BMC migration into the PDL. The IHC revealed that GFPpositive cells were detected in the PDL tissues. A number of GFP-positive cells appeared on mechanically loaded periodontal tissue, especially on the tension side of the experimental group. On the other hand, little GFP-positive cells appeared in the control group. From the above results, we analyzed how they differ from the experimental group and control group [25]. Thus, these data indicated that orthodontic mechanical stress acts as a possible promoting factor of transplanted bone marrow-derived cell migration into periodontal tissues and of differentiation to fibroblasts [26].

On the other hand, mice transplanted with bone marrow cells of GFP transgenic mice were used to observe GFP-positive cells in dental pulp of mouse incisors, PLD, oral epithelial Langerhans cells, pulp fibroblasts, dental vascular endothelial cells, and osteoclasts. GFPpositive cells in the dental pulp are dendritic cell-like cells, and some odontoblast-like cells also showed a positive response to GFP. It is clear that BMCs have the ability to differentiate into teeth and related connective tissues. GFP-positive cells histopathologically differentiated into several cell types. Fluorescent immunohistochemistry (FIHC) and tartrate-resistant acid phosphatase (TRAP) staining techniques showed that these cells were detected as osteoclasts and macrophages. In addition, GFP-positive cells gathered in adjacent blood vessels. This data suggests that GFP-positive BMCs migrate to periodontal tissues and differentiate PDL tissue-specified cells.

strongly expressed [14]. After that, the isolation of synthesized HSP by a SDS-polyacrylamide gel electrophoresis first by *Drosophila*. Some of the main protein which was also led by heat shock method of treatment in non-salivary glandular system was regarded as HSP together. A puff of the mRNA history of HSP was copied. After that a high study is reporting that the colon bacillus kept during the environment, leaven, and a gene of other mammalian cells are able to receive a heat stress and preserve a gene more than they are concerned with HSP [14]. The representation of these HSPs is then a common phenomenon [15]. HSPs have molecular ancillary and functional antiapoptotic capacity [16, 17] from ancient times. That is, it is a protein that develops to escape cell death in harsh conditions for cell survival. Many HSPs also

**Figure 2.** Experimental schema (a), macro-view (b, c), and histology (d). Quotation alteration of literature #5.

96 Histology

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 induces GFP-positive transplanted BMCs into the PDL tissues.

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 approach to tooth and jaw bone regeneration.

> 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

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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

studied [36, 37].

histopathology and IHC results.

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