Influence of orthodontic forces on the distribution of proteoglycans in rat

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1 J Med Dent Sci 2003; 50: Original Article Influence of orthodontic forces on the distribution of proteoglycans in rat hypofunctional periodontal ligament Mayumi Esashika 1, Sawa Kaneko 1, Masaki Yanagishita 2 and Kunimichi Soma 1 1) Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo Japan 2) Biochemistry, Department of Hard Tissue Engineering, Division of Bio-matrix, Graduate School, Tokyo Medical and Dental University, Tokyo Japan During orthodontic treatment, it is often necessary to move the hypofunctional teeth. In this study, we revealed an influence of orthodontic forces in the hypofunctional periodontal ligament, and focused on the distribution of proteoglycans, major extracellular matrix molecules. Five-week-old rats were divided into normal group and hypofunctional group. To induce occlusal hypofunction, occluding teeth of the mandibular first molar were extracted. At 8-week-old, orthodontic force by 15 or 2 gf titanium-nickel alloy closed coil spring was applied to the mandibular first molar toward the mesial direction. Immunohistochemical analysis was performed using antibodies for chondroitin sulfate (CS) and heparan sulfate (HS). In normal group, CS was observed throughout the extracellular matrix, while HS was observed on the endothelial cells and the osteoclastic cells on compressive side. In hypofunctional group without orthodontic appliance, CS and HS were detected in less amounts. With 15 gf, CS was observed at the compressive area where no cells and fibers were present, and HS was observed at the periphery of this area. With 2 gf, however, the distribution of CS Corresponding Author: Sawa Kaneko All correspondence should be addressed to, Sawa Kaneko, D.D.S., Ph.D., Orthodontic Science Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo , Japan TEL: FAX: s.kaneko.orts@tmd.ac.jp Received February 4; Accepted March 20, 2003 and HS was similar to the normal control. These findings indicate that CS and HS were affected by orthodontic forces, and suggest their distinct functions in tissue remodeling. Key words: occlusal hypofunction, periodontal ligament, orthodontic force, proteoglycan Introduction In clinical orthodontics, it is often necessary to move hypofunctional teeth, such as open bite incisors, high-positioned canines and teeth bucco-lingual malaligned teeth. The effects of occlusal hypofunction have been studied extensively 1-9. These studies indicated that occlusal hypofunction resulted in elongation of the tooth 1, atrophic changes of the periodontal ligament, including narrowing of the periodontal space, and decrease in both the amount of periodontal fibers 2, and the number of blood vessels 3. In the periodontal ligament, different reactions may be brought about by orthodontic force between hypofunctional and normal tooth. Therefore, it is important to elucidate these differences in orthodontics. However, there have been few reports on moving hypofunctional teeth 10. Meanwhile, it has been reported that orthodontic forces with different strength resulted in distinct tissue responses; with a heavy force, tissue with no cells and fibers appeared in the periodontal ligament 11, while with a light force, tissue with no cells and

2 184 M. ESASHIKA et al. J Med Dent Sci Table 1. Outline of experiment fibers was not observed in the periodontal ligament and tooth movement was closely related with physiological movement 12,13. However, there has been no report that compared responses of periodontal ligament in moving a hypofunctional tooth by forces with different strength. Multitude of factors regulates cell functions in the extracellular matrix. Cell functions are influenced by molecules present in the extracellular matrix or they, in turn, influence on the molecular organization of the extracellular matrix. Thus, cells promote their recruitment, migration, proliferation and regulation of differentiation within the context of extracellular matrix 14. Proteoglycans, one of the major molecules in the extracellular matrix, are composed of a central core protein and one or more covalently-bound long chains of glycosaminoglycans. They are thought to contribute in withstanding the mechanical stress and are necessary in the remodeling of the tissue 15,16. In the periodontal ligament, proteoglycan contents are also thought to change in response to the mechanical stress 4,17,18. In particular, it is reported that occlusal hypofunction causes a significant decrease in CS and HS in the periodontal ligament, and suggested CS and HS are closely related to occlusal force 4. When the mechanical stress such as orthodontic force is applied, remodeling of the periodontal ligament induces cell migration, proliferation and differentiation. Consequently, the distribution of proteoglycans is considered to change in response to orthodontic forces. The purpose of this study is to clarify the changes of extracellular matrix which occur in response to orthodontic forces of normal and hypofunctional periodontal ligament, and moreover to clarify the responses of hypofunctional periodontal ligament to orthodontic forces with different strength. Materials and Methods Animals and procedure for moving tooth The animal-use protocol form conforming to the NIH guidelines as stated in the Principles of Laboratory Care (NIH Guidelines, 1985) was reviewed and approved by the Screening Committee for Animal Research of the Tokyo Medical and Dental University prior to the study. Five-wk-old, male Wistar rats were used. The rats were divided into normal groups and hypofunctional groups. Normal groups consist of a control group under normal occlusion (Norm-C) and a 15 gf experimental group under normal occlusion (Norm-15gf). Hypofunctional groups consist of a control group under occlusal hypofunction (Hypo-C), a 15 gf experimental group under occlusal hypofunction (Hypo- 15gf) and a 2 gf experimental group under occlusal hypofunction (Hypo-2gf) (Table 1). Each group consisted of 4 or more rats. Hypofunctional groups were prepared as follows. In order to induce occlusal hypofunction, the first, second and third right maxillary molars were extracted with a forceps under intraperitoneal anesthesia with ketamine hydrochloride (Sankyo Yell Co. Ltd., Tokyo,

3 PROTEOGLYCANS IN THE PERIODONTAL LIGAMENT 185 Japan) containing 20% xylazine hydrochloride (Bayer-Japan Co. Ltd., Tokyo, Japan) as a muscle relaxant (0.1 ml/100 g body weight), after anesthetization with diethyl ether. After surgery, rats were fed normally for 3 wks. At the age of 8 wks, in order to attach the titanium-nickel alloy (Ti-Ni) closed coil spring (material from Furukawa Electric Co. Ltd., Tokyo, Japan was made into coil form by Tomy International, Tokyo, Japan), the enamel tissues around the mesial, cervical side of the mandibular right first molar and the cervical side of the mandibular incisors were etched with 65% phosphoric acid (Sun Medical Co. Ltd., Shiga, Tokyo) under intraperitoneal anesthesia. Then 15 gf or 2 gf Ti-Ni closed coil spring was fixed to the etched regions with light-cure resin (Kuraray Co. Ltd., Osaka, Japan) (Hypo-15gf, Hypo-2gf). Orthodontic force was applied to the mandibular right first molar toward the mesial direction (Fig. 1). Rats without appliance were used for a hypofunctional control group (Hypo-C). Normal groups were prepared as follows. Eight-wkold untreated rats were fixed with 15 gf Ti-Ni closed coil spring in the same way as the hypofunctional groups (Norm-15gf). Rats without appliance were used for a normal control group (Norm-C). At 2, 7 d after fixing the Ti-Ni closed coil springs, rats were anesthetized with diethyl ether and sacrificed by cervical dislocation. Their mandibles were immediately removed. Preparation of histological sections The removed mandibular specimen were immediately immersed in 10% formalin in phosphate buffered saline (PBS) ph 7.0 (WAKO Pure Chemical, Osaka, Japan), as a fixative at 4 C for 1 d, decalcified in a 10% EDTA solution, ph 7.4 at 4 C for 5 wks, and finally embedded in paraffin by a conventional method. Sections of 4 Òm thickness were cut perpendicular to the longitudinal axis of the distal root of the mandibular first molar and parallel to the long axis of the root (Fig. 2), and mounted on glass slides coated with poly-llysine (Matsunami Glass Ind. Ltd., Osaka, Japan). The sections were used for hematoxylin and eosin staining (WAKO Pure Chemical) and immunohistochemical staining for chondroitin sulfate and heparan sulfate. Figure 1. Schematic drawing of appliance used in this study. Ti-Ni closed coil spring was fixed between the mandibular right first molar and the incisors. Figure 2. Schematic drawing of area for microscopic observation. a: The schematic drawing of a horizontal section of the mandibular right first molar. b: The schematic drawing of a sagittal section of the mandibular first molar. Sagittal section was cut along the horizontal line in panel a. The periodontal ligament of the distal root the area of Òm from the furcation at the mesial area as indicated by a box was selected for observation. In some specimen, the area of Òm from the cervical of tooth at the distal area was observed as distal side. Abbreviations: M1, mandibular first molar; M, mesial side; D, distal side; arrow, direction of orthodontic force. Table 2. Monoclonal antibodies used in the present study Immunohistochemistry for chondroitin sulfate and heparan sulfate Characteristics of the antibodies used in the present study are summarized in Table 2. Deparaffinized sec-

4 186 M. ESASHIKA et al. J Med Dent Sci tions were immersed in methanol (WAKO Pure Chemical) containing 0.3% hydrogen peroxide (WAKO Pure Chemical) for 30 min to block endogenous peroxidase activity, washed 3 times in 0.01 M PBS at ph 7.2, and exposed to normal rabbit serum (Nichirei, Tokyo, Japan) for 20 min, followed by reaction with primary antibodies overnight in humidified chambers at 4 C at concentrations described in Table 2. Excess antibodies were removed by washing with PBS 3 times, and the bound antibodies were detected with biotinylated rabbit anti-mouse immunoglobulin (Nichirei), and peroxidase conjugated streptavidin (Nichirei). After 3 additional washes with PBS, bound peroxidase was visualized with 0.02% diaminobenzidine (Sigma, St. Louis, MO, USA) in 0.05 M Tris buffer at ph 7.6 plus 0.02% hydrogen peroxide. The sections were rinsed in distilled water, dehydrated in ascending concentrations of ethanol, cleared in xylene, and mounted with xylene (Daido Sangyo Co. Ltd., Tokyo, Japan). Counterstaining was performed using hematoxylin (WAKO Pure Chemical). As negative controls for primary antibodies, PBS was used in place of antibodies. Both control and experimental slides were processed at the same time, and staining conditions such as incubation time of primary antibodies and diaminobenzidine were identical. Variation of hematoxylin and eosin staining and immunohistochemistry among 4 or more rats in the same experimental group was generally small. Negative control specimens omitted the primary antibody did not show any significant non-specific staining. Area for observation The periodontal ligament of the distal root the area of Òm from the furcation at the mesial area as indicated by a box was selected for observation (Fig. 2). This area is regarded as a compressive side in orthodontic tooth movement 13,19. And in some specimen, the area of Òm from the cervical of tooth at the distal area of the distal root was observed as distal side. Results The histological and immunohistochemical findings of the periodontal ligament are summarized in Table 3. General histological findings In the control group under normal occlusion (Norm- C) sample, the fibrous network structures were observed. Fibroblasts were arranged among the fibers (Fig. 3a). In the compressive side of 2 d of experimental group under normal occlusion (Norm-15gf) sample, the arrangement of fibers became longitudinal and regular. The periodontal space was narrowed (Fig. 3b). In the 7 d of Norm-15gf sample, the arrangement of fibrous structure remained regular. The width of the periodontal ligament regained its normal width (Fig. 3c). In the control group under hypofunction (Hypo-C) sample, significant narrowing of the periodontal space was observed. The fibrous network structures were Table 3-a. Summarys of immunohistochemical findings of the periodontal ligament for chondroitin sulfate Table 3-b. Summarys of immunohistochemical findings of the periodontal ligament for heparan sulfate in mesial side. Table 3-b. Some sections observed at distal side shows in a parenthesis.

5 PROTEOGLYCANS IN THE PERIODONTAL LIGAMENT 187 Figure 3. Sections showing hematoxylin and eosin staining at the mesial side of the distal root of mandibular right first molar. Panels: a, Norm-C; b, 2 d of Norm-15gf; c, 7 d of Norm-15gf; d, Hypo-C; e, 2 d of Hypo-15gf; f, 7 d of Hypo-15gf; g, 2 d of Hypo-2gf; h, 7 d of Hypo-2gf. In the Norm-C sample, the fibrous network structures of the periodontal ligament were observed (a). In the Hypo-C sample, the significant narrowing of the periodontal space was observed. The fibrous network structures disappeared (d). When tooth was applied with 15 gf, the area with no cells and fibers was observed (e,f). When tooth was applied with 2 gf, the fibrous structures of the periodontal ligament were dense and regular. Some microvasculatures were observed (h). Abbreviations: B, alveolar bone; PDL, periodontal ligament; R, root; Bar = 100 Òm.

6 188 M. ESASHIKA et al. J Med Dent Sci drastically reduced in comparison with the Norm-C sample. The number of microvasculatures was decreased throughout the periodontal ligament (Fig. 3d). In the compressive side of the 2 d of Hypo-15gf sample, the periodontal space was further narrowed. Area with no cells and fibrous structures was noticeable (Fig. 3e). In the 7 d of Hypo-15gf sample, the periodontal space was further narrowed. And the area with no cells and fibers increased in the compressive side (Fig. 3f). In the compressive side of 2 d of Hypo-2gf sample, the periodontal space was narrowed in comparison with the Hypo-C sample. Fibroblasts in the compressive side decreased in number and were arranged more irregularly than those in the Hypo-C sample. The fibrous structures were disarranged (Fig. 3g). In the 7 d of Hypo-2gf sample, the periodontal space recovered. The arranged fibrous structures became dense and regular. Some microvasculatures were observed (Fig. 3h). Immunohistochemical localization of chondroitin sulfate In the Norm-C sample, a stronger positive reaction for chondroitin sulfate (CS) was easily observed on the cervical side than on the apical side in the periodontal ligament at a low magnification (data not shown, it is also described at the other paper 4 ). At a high magnification of the Norm-C sample, CS was homogeneously observed in the extracellular matrix, with some emphasis along the fibrous structures and around the vasculatures (Fig. 4a). In the 2 d and 7 d of Norm-15gf samples, CS showed little difference from that of the Norm-C sample (Figs. 4b, c). In the Hypo-C sample, reactivity for CS showed a significant and uniform decrease in the extracellular matrix in comparison with the Norm-C sample (Fig. 4d). CS in the compressive side of the 2 d of Hypo-15gf sample was homogeneously observed in the extracellular matrix and more intense than the Hypo-C sample (Fig. 4e). In the 7 d of Hypo-15gf sample, immunoreactivity for CS was more intense. CS was observed in the extracellular matrix homogeneously, with some emphasis in the areas with no visible cells and fibers (Fig. 4f). In the compressive side of the 2 d of Hypo-2gf sample, reaction for CS slightly increased in the extracellular matrix in comparison with the Hypo-C sample (Fig. 4g). In the 7 d of Hypo-2gf sample, reaction for CS was more intense. CS was homogeneously observed in the extracellular matrix along the fibrous structures, and around the microvasculatures (Fig. 4h). Immunohistochemical localization of heparan sulfate In the periodontal ligament of the Norm-C sample, a positive reaction for heparan sulfate (HS) at the distal side generally showed stronger reactions than that at the mesial side at a low magnification (Fig. 5a). At a high magnification of the distal side, HS was mostly located on the cell surface of the vascular endothelial cells and of the osteoclastic cells (Fig. 7a). In the mesial side, HS was mostly seen on the cell surface of the vascular endothelial cells, however the number of the microvasculatures was fewer than that in the distal side (Fig. 6a). In the 2 d of Norm-15gf sample, reactivity for HS in the compressive side was more intense than in the Norm-C sample at a low magnification (Fig. 5b). At a high magnification, HS in the compressive side was located on the cell surface of the vascular endothelial cells (Fig. 6b). In the 7 d of Norm-15gf specimen, positive reaction for HS was observed in the extracellular matrix of the periodontal ligament in the compressive side at a low magnification. The reaction for HS on this side was more intense than that for 2 d of Norm-15gf sample (Fig. 5c). At a high magnification, HS was observed on the cell surface of the osteoclastic cells surrounding the alveolar bone, and of the vascular endothelial cells. Positive reaction for HS was also observed in the extracellular matrix in compressive side (Fig. 6c). Immunoreaction for HS of the Hypo-C sample showed a significant decrease throughout the extracellular matrix in comparison with that of the Norm-C specimen (Fig. 6d). In the compressive side of the 2 d of Hypo-15gf sample, a weakly positive reaction was observed (Fig. 6e). In the 7 d of Hypo-15gf sample, a mildly positive reaction for HS was observed in the extracellular matrix, especially the peripheral area with no visible cells and fibers (Fig. 6f). In the compressive side of the 2 d of Hypo-2gf, microvasculatures with small diameter were observed and the immunoreactivity for HS was observed on the cell surface of the endothelial cells (Fig. 6g). In the 7 d of Hypo-2gf sample, reaction for HS in the compressive side was intense on the vascular endothelial cells and the osteoclastic cells surrounding the alveolar bone of the periodontal ligament. The diameter and the number of microvasculatures stained for HS increased from 2 d of Hypo-2gf sample (Fig. 6h). The distal side of the periodontal ligament showed a reaction for HS on the vascular endothelial cells. The diameter of microvas-

7 PROTEOGLYCANS IN THE PERIODONTAL LIGAMENT 189 Figure 4. Sections showing immunostaining for chondroitin sulfate (CS) at the mesial side of the distal root of mandibular right first molar. Panels: a, Norm-C; b, 2 d of Norm-15gf; c, 7 d of Norm-15gf; d, Hypo-C; e, 2 d of Hypo-15gf; f, 7 d of Hypo-15gf; g, 2 d of Hypo-2gf; h, 7 d of Hypo-2gf. CS was homogeneously observed in the extracellular matrix, with some emphasis along the fibrous structures and around the vasculatures (a-c). In the Hypo-C sample, CS decreased significantly in comparison with Norm-C sample (d). When tooth was applied with 15 gf, CS was observed at the area with no cells and fibers (e,f). With 2 gf, the distribution of CS was observed around microvasculatures (arrow heads in panel h) and fibrous structure. The distribution of CS was similar to normal control (h). Abbreviations: B, alveolar bone; PDL, periodontal ligament; R, root; Bar = 100 Òm.

8 190 M. ESASHIKA et al. J Med Dent Sci Figure 5. Sections showing immunostaining for heparan sulfate (HS) in normal group of distal root at low magnification (a-c). Panels: a, Norm-C; b, 2 d of Norm-15gf; c, 7 d of Norm-15gf. A positive reaction for HS at the distal side generally showed stronger reactions than that at the mesial side (a). Reactivity for HS in the compressive mesial side was more intense than in the Norm-C sample (b). In the 7 d of Norm-15gf sample, the reaction for HS was more intense than that in the 2 d of Norm-15gf sample (c). Abbreviations: M1, mandibular first molar; M, mesial side; D, distal side; Bar = 500 Òm. culature in the distal side was smaller than that in the compressive side. The number of microvasculatures in the distal side was fewer than that in compressive side (Fig. 7b). Discussion The load of the Ti-Ni closed coil spring that we used in the present study did not diminish appreciably as displacement of tooth increased. Therefore, we could evaluate the response of the periodontal ligament to the continuous orthodontic forces with different strength. It is found that the narrowing of microvasculatures could not be brought about with less than 80 g/cm 2 in human 20. This pressure would be approximately equivalent to 10 gf in the rat maxillary first molar 21. Therefore, we selected 15 gf of orthodontic force as a slightly stronger force in order not to obtain similar observations between under normal occlusion and occlusal hypofunction. And for hypofunctional teeth, we selected 2 gf as a light force. There are many reports on the atrophic changes in the periodontal ligament under occlusal hypofunction 1-9. It is reported that, at 2 wks 5 or 3 wks 9 under hypofunction, the structures of the periodontal ligament were disorganized and the arrangement of fibers became parallel to the alveolar walls. And at 4 wks and 12 wks under occlusal hypofunction, the condition of the periodontal ligament did not appear different from that of the 2 or 3 wks 5. Therefore, we chose to begin loading the orthodontic force after 3 wks post extraction of the maxillary molars. And we successfully induced atrophic changes in the periodontal ligament. It is generally considered that orthodontic tooth movement consists of clearly separatable three phases occurring in the periodontal ligament: an initial compressive phase consisting of changes in the visco-elasticity of periodontal ligament for 1-4d; a follow-up lag phase in which the tooth movement slows down for 4-7d with the appearance of the tissue with no cells and fibers in the periodontal ligament; and finally a phase in which the tooth moves progressively in association with bone resorption for 7-14d. It is concluded that with a 40 gf in rats, the second phase was so short that the tooth movement in association with bone resorption had occurred by 7d 11. So, we made observation on 2 d as initial phase and 7 d as second or third phase in order

9 PROTEOGLYCANS IN THE PERIODONTAL LIGAMENT 191 Figure 6. Sections showing immunostaining for heparan sulfate (HS) at the mesial side of the distal root of mandibular right first molar. Panels: a, Norm-C; b, 2 d of Norm-15gf; c, 7 d of Norm-15gf; d, Hypo-C; e, 2 d of Hypo-15gf; f, 7 d of Hypo-15gf; g, 2 d of Hypo-2gf; h, 7 d of Hypo-2gf. HS was seen on the cell surface of the vascular endothelial cells (a,b). HS was observed on the cell surface of the osteoclastic cells surrounding the alveolar bone and of the vascular endothelial cells (c). In the Hypo-C sample, HS decreased significantly in comparison with Norm-C sample (d). When tooth was applied with 15 gf, HS was observed at the peripheral area with no cells and fibers (e,f). With 2 gf, the distribution of HS was observed on the endothelial cells (arrow heads in panel g and h) and the osteoclastic cells (arrow in panel h). The distribution of HS was similar to normal control (h). Abbreviations: B, alveolar bone; PDL, periodontal ligament; R: root; Bar = 100 Òm.

10 192 M. ESASHIKA et al. J Med Dent Sci Figure 7. Sections showing immunostaining for heparan sulfate (HS) in distal side of the distal root of mandibular right first molar. Panels: a, Norm-C; b, 7 d of Hypo-2gf. HS was observed on the endothelial cells (arrow heads in panel a) and the osteoclastic cells in distal side (arrow in panel a). The number of the microvasculatures was more than that in the mesial side (a). A reaction for HS on the vascular endothelial cells was observed. The number of microvasculatures in the distal side was fewer than that in mesial side. The diameter of microvasculature in the distal side was smaller than that in the mesial side (b). Abbreviations: B, alveolar bone; PDL, periodontal ligament; R, root; Bar = 100 Òm. to evaluate the distinctive remodeling for orthodontic force. We used CS56 antibody to detect chondroitin sulfate, and 10E4 antibody to detect heparan sulfate. CS56 antibody specifically binds to Domain 2 of chondroitin sulfate E4 antibody specifically binds to N-unsubstituted glucosamine of heparan sulfate 23. In the normal periodontal ligament, the components of the periodontal ligament such as the collagen fibers and the endothelial cells were arranged to resist occlusal force and mastication force. When orthodontic force was applied to the normal periodontal ligament, the distribution of CS did not change significantly. Immunoreactivity for CS is intense in normal control, and we might be not able to detect the influence of orthodontic force by immunohistochemical analysis. In the hypofunctional periodontal ligament, the fibers became disarranged and the number of blood vessels decreased. In the Hypo-C sample, immunostaining for CS was weak with atrophic changes of periodontal ligament, similar to a previous report 4. When orthodontic force of 15 gf was applied to the hypofunctional periodontal ligament, the area with no cells and fibers was observed. In the normal periodontal ligament, it is reported that, the area with no cells and fibers so-called a hyalinized tissue was observed at the over-compressed area with 40 gf 11 or 50 gf 24 of orthodontic force in rat. And multi-nucleated cells have been observed at the margin of the hyalinized tissue, and are speculated to resorb this tissue. The hyalinized tissue was replaced to a new tissue. We can define a hyalinized tissue. It is reported that chondroitin 6-sulfate was present at this area and suggested that distribution of chondroitin 6-sulfate was related to the compressive force in the periodontal ligament 17. In this study, the immunoreactivity for CS in Hypo-15gf sample was the same result. Thus, CS may react to orthodontic force and participate in tissue remodeling. However, it may be that cells disappeared for some mechanism, and extracellular matrix remained. Or it may be that hyalinized tissue have formed far from the origin where it was synthesized. Since CS distribution of Hypo-15gf is not clear, the further study is required. When orthodontic force of 2 gf was applied to the hypofunctional periodontal ligament, positive reaction for CS was observed along the fibers in the 7 d sample. It is reported that mechanical stress increases collagen synthesis 25. Therefore, orthodontic force of 2 gf may have increased the amount of collagen fibers. Meanwhile, it was reported that decorin, one of the CS proteoglycans which binds to type I collagen regulates their fiber formation 26,27. It was also reported that decorin increases the diameter of collagen fibers 28. Orthodontic forces might have reacted to collagen and CS synthesis and CS participate in reconstruction of collagen fibers. HS is found to be associated with various biological processes of growth factors and cytokines, as well as being implicated in cell adhesion, recognition, and migration. It is reported that the shear stress in blood flow increases synthesis of HS in vascular endothelial cells in vitro 29. And it is reported that HS may act together with shear stress to modify and maintain the endothelial cell configuration 30. When mechanical stress increases the expression of HS and regulates several growth factors, the remodeling system of vascular wall may have been induced 31. In the Norm-C, the 2 d of Norm-15gf and the 7 d of Norm-15gf samples, there were vascular endothelial cells stained for HS at a high magnification. HS on the endothelial cell may be interpreted to participate in vascular remodeling. In the distal side of Norm-C and the compressive side of 7 d of Norm-15gf sample, HS was detected on the cell surface of the osteoclastic cells surrounding the alveolar bone. It is reported that HS binds cytokines and protect from the decomposition and inactivation, and participated in differentiation and activation of osteoclast 32. Addition of heparin or HS induces increased bone resorption by osteoclasts in vitro 33.

11 PROTEOGLYCANS IN THE PERIODONTAL LIGAMENT 193 Under the normal condition in rat, as first molar physiologically moves toward the distal side, this side becomes compressed 34,35. However, in the present study, as the orthodontic force was applied to the first molar toward the mesial direction, mesial side becomes compressed. The HS positive area was situated at compressed area. HS expressed on the cell surface of osteoclastic cells may be related to the bone resorption mechanism. When orthodontic force of 15 gf was applied to the hypofunctional periodontal ligament, HS was detected in the peripheral area with no cells and fibers. It is reported that multi-nucleated cells have been observed at the margin of the tissue with no cells and fibers and are speculated to absorb the tissue in orthodontically over-compressed zones 25,36. HS may take part in the remodeling of the tissue with no cells and fibers. When orthodontic force of 2 gf was applied to the hypofunctional periodontal ligament, the diameter of microvasculature in the 7 d sample increased in comparison with that in the 2 d sample. It is reported that an increase in blood flow causes the vessels dilate 37. In the Hypo-2gf samples, orthodontic light force may have increased the shear stress in blood flow of periodontal ligament. The present study suggested that the application of the orthodontic force to hypofunctional tooth is different from that to normal tooth. And when forces with different strength were applied to hypofunctional periodontal ligament, different reaction occurred. Distinct functions of CS and HS participating in tissue remodeling in response to the orthodontic force in the periodontal ligament may be performed. However, to fully understand the functions of proteoglycan in tissue remodeling caused by orthodontic forces, further study such as immunohistochemistry for core proteins is required. References 1. Ohshima S, Komatsu K, Yamane A, et al. Prolonged effects of hypofunction on the mechanical strength of the periodontal ligament in rat mandibular molars. Arch Oral Biol 1991;36: Kinoshita Y. Tonooka K. Chiba M. The effect of hypofunction on the mechanical properties of the periodontium in the rat mandibular first molar. Arch Oral Biol 1982;27: Tanaka A, Iida J, Soma K. Effect of hypofunction on the microvasculature in the periodontal ligament of the rat molar. J Jpn Orthod Soc 1998;57: Kaneko S, Ohashi K, Soma K, et al. Occlusal hypofunction causes changes of proteoglycan content in the rat periodontal ligament. J Periodontal Res 2001;36: Levy GG, Mailland ML. Histologic study of the effects of occlusal hypofunction following antagonist tooth extraction in the rat. J Periodontol 1980;5: Short E. Effects of tooth function on adjacent alveolar bone and Sharpey s fibers of the rat periodontium. Anat Rec 1990;227: Eui-Seok Suhr, Warita H, Iida J, et al. The effects of occlusal hypofunction and its recovery on the periodontal tissues of the rat molar: ED1 immunohistochemical study. Orthodontic Waves 2002;61: Hayashi Y, Iida J, Warita H, et al. Effects of occlusal hypofunction on the microvasculature and endothelin expression in the periodontal ligaments of rat molars. Orthodontic Waves 2001;60: Koike K. The effects of loss and restoration of occlusal function on the periodontal tissues of rat molar teeth Histopathological and histometrical investigation. J Jpn Soc Periodont 1996;38: Lee Minyeong, Nakamura Y. A histological study on the periodontal ligament during the experimental movement of hypofunctional teeth in rats On the tension side. Orthodontic Waves 1999;58: King GJ, Fischlschweiger W. The effect of force magnitude on extractable bone resorptive activity and cemental cratering in orthodontic tooth movement. J Dent Res 1982;61: Kohno T, Matsumoto Y, Kanno Z, et al. Experimental tooth movement under light orthodontic forces: rates of tooth movement and changes of the periodontium. J Orthod 2002;29: Warita H, Iida J, Yamaguchi S, et al. A study on experimental tooth movement with Ti-Ni alloy orthodontic wires: Comparison between light continuous force and light dissipating force. J Jpn Orthod Soc 1996;55: Adams JC, Watt FM. Regulation of development and differentiation by the extracellular matrix. Development 1993;117: Schneiderman R, Keret D, Maroudas A. Effects of mechanical and osmotic pressure on the rate of glycosaminoglycan synthesis in the human adult femoral head cartilage: an in vitro study. J Orthop Res 1986;4: Echtermeyer F, Baciu PC, Saoncella S, et al. Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers. J Cell Sci 1999;112: Kagayama M, Sasano Y, Mizoguchi I, et al. Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement. J Periodontal Res 1996;3: Sato R, Yamamoto H, Kasai K, et al. Distribution pattern of versican, link protein and hyaluronic acid in the rat periodontal ligament during experimental tooth movement. J Periodontal Res 2002;37: Azuma M. Study on histologic changes of periodontal membrane incident to experimental tooth movement. Bull Tokyo Med Dent Univ 1970;17: Kondo K. A study of blood circulation in the periodontal membrane by electrical impedance plethysmography. J Stomatol Soc Jpn 1969;36: Sato T, Iida J, Kurihara S. A study of tooth movement with super-elastic force. J Jpn Orthod Soc 1984;43: Avnur Z, Geiger B. Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody. Cell 1984;38: David G, Bai XM, Van der Schueren B, et al. Developmental changes in heparan sulfate expression: in situ detection with mabs. J Cell Biol 1992;119:

12 194 M. ESASHIKA et al. J Med Dent Sci 24. Brudvik P, Rygh P. Multi-nucleated cells remove the main hyalinized tissue and start resorption of adjacent root surfaces. Eur J Orthod 1994;16: Robbins JR, Evanko SP, Vogel KG. Mechanical loading and TGF-beta regulate proteoglycan synthesis in tendon. Arch Biochem Biophys 1997;342: Scott JE. The structure of interfibrillar proteoglycan bridges (shape modules) in extracellular matrix of fibrous connective tissues and their stability in various chemical environments. J Anat 1998;192: Schonherr E, Hausser H, Beavan L, et al. Decorin-type I collagen interaction. Presence of separate core protein-binding domains. J Biol Chem 1995;270: Kuc IM, Scott PG. Increased diameters of collagen fibrils precipitated in vitro in the presence of decorin from various connective tissues. Connect Tissue Res 1997;36: Arisaka T, Mitsumata M, Kawasumi M, et al. Effects of shear stress on glycosaminoglycan synthesis in vascular endothelial cells. Ann N Y Acad Sci 1995;17: Ookawa K, Sato M, Ohshima N. Morphological changes of endothelial cells after exposure to fluid-imposed shear stress: differential responses induced by extracellular matrices. Biorheology 1993;30: Worapamorn W, Li H, Haas HR, et al. Cell surface proteoglycan expression by human periodontal cells. Connect Tissue Res 2000;4: Nakamura H. Morphological study on cell-cell interaction between osteoclasts and osteoblasts. (in Japanese). Kaibogaku Zasshi 2000;75: Chowdhury MH, Hamada C, Dempster DW. Effects of heparin on osteoclast activity. J Bone Miner Res 1992;7: Worapamorn W, Haase HR, Li H, et al. Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells. J Cell Physiol 2001;186: Roux D, Woda A. Biometric analysis of tooth migration after approximal contact removal in the rat. Arch Oral Biol 1994;39: Miura F, Inoue N, Azuma M, et al. Development and organization of periodontal membrane and physiologic tooth movement. Bull Tokyo Med Dent Univ 1970;17: Rygh P. Orthodontic root resorption studied by electron microscopy. Angle Orthod 1977;47: Siegel G, Walter A, Kauschmann A, et al. Anionic biopolymers as blood flow sensors. Biosens Bioelectron 1996;11: