inﬂuence of orthodontic forces on the distribution of...

During orthodontic treatment, it is often neces-sary to move the hypofunctional teeth. In thisstudy, we revealed an influence of orthodonticforces in the hypofunctional periodontal ligament,and focused on the distribution of proteoglycans,major extracellular matrix molecules. Five-week-oldrats were divided into normal group and hypo-functional group. To induce occlusal hypofunction,occluding teeth of the mandibular first molar wereextracted. At 8-week-old, orthodontic force by 15 or2 gf titanium-nickel alloy closed coil spring wasapplied to the mandibular first molar toward themesial direction. Immunohistochemical analysiswas performed using antibodies for chondroitinsulfate (CS) and heparan sulfate (HS). In normalgroup, CS was observed throughout the extracel-lular matrix, while HS was observed on theendothelial cells and the osteoclastic cells oncompressive side. In hypofunctional group withoutorthodontic appliance, CS and HS were detected inless amounts. With 15 gf, CS was observed at thecompressive area where no cells and fibers werepresent, and HS was observed at the periphery ofthis area. With 2 gf, however, the distribution of CS

and HS was similar to the normal control. Thesefindings indicate that CS and HS were affected byorthodontic forces, and suggest their distinctfunctions in tissue remodeling.

Key words: occlusal hypofunction, periodontal lig-ament, orthodontic force, proteogly-can

Introduction

In clinical orthodontics, it is often necessary tomove hypofunctional teeth, such as open biteincisors, high-positioned canines and teeth bucco-lin-gual malaligned teeth. The effects of occlusal hypo-function have been studied extensively1-9. These stud-ies indicated that occlusal hypofunction resulted in elon-gation of the tooth1, atrophic changes of theperiodontal ligament, including narrowing of the peri-odontal space, and decrease in both the amount ofperiodontal fibers2, and the number of blood vessels3.In the periodontal ligament, different reactions may bebrought about by orthodontic force between hypo-functional and normal tooth. Therefore, it is important toelucidate these differences in orthodontics. However,there have been few reports on moving hypofunctionalteeth10. Meanwhile, it has been reported that ortho-dontic forces with different strength resulted in distincttissue responses; with a heavy force, tissue with nocells and fibers appeared in the periodontal liga-ment11, while with a light force, tissue with no cells and

Original Article

Influence of orthodontic forces on the distribution of proteoglycans in rat hypofunctional periodontal ligament

Mayumi Esashika1, Sawa Kaneko1, Masaki Yanagishita2 and Kunimichi Soma1

1) Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences,Graduate School, Tokyo Medical and Dental University, Tokyo Japan2) Biochemistry, Department of Hard Tissue Engineering, Division of Bio-matrix, Graduate School, TokyoMedical and Dental University, Tokyo Japan

J Med Dent Sci 2003; 50: 183–194

Corresponding Author: Sawa KanekoAll correspondence should be addressed to, Sawa Kaneko, D.D.S.,Ph.D., Orthodontic Science Department of Orofacial Developmentand Function, Division of Oral Health Sciences, Graduate School,Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,Tokyo 113-8549, JapanTEL: +81-3-5803-5529 FAX: +81-3-5803-5530E-mail: [email protected] February 4; Accepted March 20, 2003

fibers was not observed in the periodontal ligament andtooth movement was closely related with physiologicalmovement12,13. However, there has been no reportthat compared responses of periodontal ligament inmoving a hypofunctional tooth by forces with differentstrength.

Multitude of factors regulates cell functions in theextracellular matrix. Cell functions are influenced bymolecules present in the extracellular matrix or they, inturn, influence on the molecular organization of theextracellular matrix. Thus, cells promote their recruit-ment, migration, proliferation and regulation of differ-entiation within the context of extracellular matrix14.Proteoglycans, one of the major molecules in theextracellular matrix, are composed of a central coreprotein and one or more covalently-bound long chainsof glycosaminoglycans. They are thought to con-tribute in withstanding the mechanical stress and arenecessary in the remodeling of the tissue15,16. In theperiodontal ligament, proteoglycan contents are alsothought to change in response to the mechanicalstress4,17,18. In particular, it is reported that occlusalhypofunction causes a significant decrease in CS andHS in the periodontal ligament, and suggested CS andHS are closely related to occlusal force4. When themechanical stress such as orthodontic force isapplied, remodeling of the periodontal ligamentinduces cell migration, proliferation and differentiation.Consequently, the distribution of proteoglycans isconsidered to change in response to orthodonticforces.

The purpose of this study is to clarify the changes of

extracellular matrix which occur in response to ortho-dontic forces of normal and hypofunctional periodontalligament, and moreover to clarify the responses ofhypofunctional periodontal ligament to orthodonticforces 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 ofLaboratory Care’ (NIH Guidelines, 1985) wasreviewed and approved by the Screening Committeefor Animal Research of the Tokyo Medical and DentalUniversity prior to the study.

Five-wk-old, male Wistar rats were used. The ratswere divided into normal groups and hypofunctionalgroups. Normal groups consist of a control groupunder normal occlusion (Norm-C) and a 15 gf experi-mental group under normal occlusion (Norm-15gf).Hypofunctional groups consist of a control groupunder occlusal hypofunction (Hypo-C), a 15 gf experi-mental group under occlusal hypofunction (Hypo-15gf) and a 2 gf experimental group under occlusalhypofunction (Hypo-2gf) (Table 1). Each group con-sisted of 4 or more rats.

Hypofunctional groups were prepared as follows. Inorder to induce occlusal hypofunction, the first, secondand third right maxillary molars were extracted with aforceps under intraperitoneal anesthesia with keta-mine hydrochloride (Sankyo Yell Co. Ltd., Tokyo,

M. ESASHIKA et al. J Med Dent Sci184

Table 1. Outline of experiment

Japan) containing 20% xylazine hydrochloride(Bayer-Japan Co. Ltd., Tokyo, Japan) as a musclerelaxant (0.1 ml/100 g body weight), after anesthetiza-tion with diethyl ether. After surgery, rats were fed nor-mally for 3 wks. At the age of 8 wks, in order to attachthe 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 thecervical side of the mandibular incisors were etchedwith 65% phosphoric acid (Sun Medical Co. Ltd.,Shiga, Tokyo) under intraperitoneal anesthesia. Then15 gf or 2 gf Ti-Ni closed coil spring was fixed to theetched regions with light-cure resin (Kuraray Co. Ltd.,Osaka, Japan) (Hypo-15gf, Hypo-2gf). Orthodonticforce was applied to the mandibular right first molartoward the mesial direction (Fig. 1). Rats withoutappliance were used for a hypofunctional controlgroup (Hypo-C).

Normal groups were prepared as follows. Eight-wk-old untreated rats were fixed with 15 gf Ti-Ni closed coilspring in the same way as the hypofunctional groups(Norm-15gf). Rats without appliance were used for anormal control group (Norm-C).

At 2, 7 d after fixing the Ti-Ni closed coil springs, ratswere anesthetized with diethyl ether and sacrificed bycervical dislocation. Their mandibles were immediatelyremoved.

Preparation of histological sections The removed mandibular specimen were immedi-

ately immersed in 10% formalin in phosphate bufferedsaline (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 finallyembedded in paraffin by a conventional method.Sections of 4 Òm thickness were cut perpendicular tothe longitudinal axis of the distal root of the mandibularfirst molar and parallel to the long axis of the root (Fig.2), and mounted on glass slides coated with poly-L-lysine (Matsunami Glass Ind. Ltd., Osaka, Japan).The sections were used for hematoxylin and eosinstaining (WAKO Pure Chemical) and immunohisto-chemical staining for chondroitin sulfate and heparansulfate.

Immunohistochemistry for chondroitin sulfateand heparan sulfate

Characteristics of the antibodies used in the presentstudy are summarized in Table 2. Deparaffinized sec-

185PROTEOGLYCANS IN THE PERIODONTAL LIGAMENT

Figure 1. Schematic drawing of appliance used in this study. Ti-Niclosed coil spring was fixed between the mandibular right firstmolar and the incisors.

Figure 2. Schematic drawing of area for microscopic observation. a:The schematic drawing of a horizontal section of the mandibular rightfirst molar. b: The schematic drawing of a sagittal section of themandibular first molar. Sagittal section was cut along the horizontalline in panel a. The periodontal ligament of the distal root the area of500-800 Òm from the furcation at the mesial area as indicated by abox was selected for observation. In some specimen, the area of 500-800 Òm from the cervical of tooth at the distal area was observed asdistal 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

tions were immersed in methanol (WAKO PureChemical) containing 0.3% hydrogen peroxide(WAKO Pure Chemical) for 30 min to block endoge-nous peroxidase activity, washed 3 times in 0.01 MPBS at pH 7.2, and exposed to normal rabbit serum(Nichirei, Tokyo, Japan) for 20 min, followed by reactionwith primary antibodies overnight in humidified cham-bers at 4°C at concentrations described in Table 2.Excess antibodies were removed by washing withPBS 3 times, and the bound antibodies were detectedwith biotinylated rabbit anti-mouse immunoglobulin(Nichirei), and peroxidase conjugated streptavidin(Nichirei). After 3 additional washes with PBS, boundperoxidase was visualized with 0.02% diaminobenzi-dine (Sigma, St. Louis, MO, USA) in 0.05 M Trisbuffer at pH 7.6 plus 0.02% hydrogen peroxide. Thesections were rinsed in distilled water, dehydrated inascending concentrations of ethanol, cleared inxylene, and mounted with xylene (Daido Sangyo Co.Ltd., Tokyo, Japan). Counterstaining was performedusing hematoxylin (WAKO Pure Chemical). As negativecontrols for primary antibodies, PBS was used inplace of antibodies. Both control and experimentalslides were processed at the same time, and stainingconditions such as incubation time of primary antibod-ies and diaminobenzidine were identical.

Variation of hematoxylin and eosin staining andimmunohistochemistry among 4 or more rats in thesame experimental group was generally small.Negative control specimens omitted the primary anti-body did not show any significant non-specific staining.

Area for observationThe periodontal ligament of the distal root the area of

500-800 Òm from the furcation at the mesial area asindicated by a box was selected for observation (Fig.2). This area is regarded as a compressive side inorthodontic tooth movement13,19. And in some speci-men, the area of 500-800 Òm from the cervical of toothat the distal area of the distal root was observed as dis-tal side.

Results

The histological and immunohistochemical findings ofthe periodontal ligament are summarized in Table 3.

General histological findingsIn the control group under normal occlusion (Norm-

C) sample, the fibrous network structures wereobserved. Fibroblasts were arranged among thefibers (Fig. 3a). In the compressive side of 2 d of exper-imental group under normal occlusion (Norm-15gf)sample, the arrangement of fibers became longitudinaland regular. The periodontal space was narrowed(Fig. 3b). In the 7 d of Norm-15gf sample, thearrangement of fibrous structure remained regular.The width of the periodontal ligament regained its nor-mal width (Fig. 3c).

In the control group under hypofunction (Hypo-C)sample, significant narrowing of the periodontal spacewas 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.


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 dof Hypo-2gf. In the Norm-C sample, the fibrous network structures of the periodontal ligament were observed (a). In the Hypo-C sam-ple, the significant narrowing of the periodontal space was observed. The fibrous network structures disappeared (d). When toothwas applied with 15 gf, the area with no cells and fibers was observed (e,f). When tooth was applied with 2 gf, the fibrous structuresof the periodontal ligament were dense and regular. Some microvasculatures were observed (h). Abbreviations: B, alveolar bone;PDL, periodontal ligament; R, root; Bar = 100 Òm.

drastically reduced in comparison with the Norm-Csample. The number of microvasculatures wasdecreased throughout the periodontal ligament (Fig.3d). In the compressive side of the 2 d of Hypo-15gfsample, 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 peri-odontal space was further narrowed. And the area withno 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 withthe Hypo-C sample. Fibroblasts in the compressiveside decreased in number and were arranged moreirregularly than those in the Hypo-C sample. Thefibrous structures were disarranged (Fig. 3g). In the 7 dof Hypo-2gf sample, the periodontal space recovered.The arranged fibrous structures became dense andregular. Some microvasculatures were observed (Fig.3h).

Immunohistochemical localization of chondroitinsulfate

In the Norm-C sample, a stronger positive reactionfor chondroitin sulfate (CS) was easily observed on thecervical side than on the apical side in the periodontalligament at a low magnification (data not shown, it isalso described at the other paper4). At a high magnifi-cation of the Norm-C sample, CS was homogeneouslyobserved in the extracellular matrix, with someemphasis along the fibrous structures and around thevasculatures (Fig. 4a). In the 2 d and 7 d of Norm-15gfsamples, CS showed little difference from that of theNorm-C sample (Figs. 4b, c).

In the Hypo-C sample, reactivity for CS showed asignificant and uniform decrease in the extracellularmatrix in comparison with the Norm-C sample (Fig. 4d).CS in the compressive side of the 2 d of Hypo-15gfsample was homogeneously observed in the extracel-lular matrix and more intense than the Hypo-C sample(Fig. 4e). In the 7 d of Hypo-15gf sample, immunore-activity for CS was more intense. CS was observed inthe extracellular matrix homogeneously, with someemphasis in the areas with no visible cells and fibers(Fig. 4f).

In the compressive side of the 2 d of Hypo-2gf sam-ple, reaction for CS slightly increased in the extracellu-lar matrix in comparison with the Hypo-C sample (Fig.4g). In the 7 d of Hypo-2gf sample, reaction for CS wasmore intense. CS was homogeneously observed in theextracellular matrix along the fibrous structures, and

around the microvasculatures (Fig. 4h).

Immunohistochemical localization of heparan sul-fate

In the periodontal ligament of the Norm-C sample, apositive reaction for heparan sulfate (HS) at the distalside generally showed stronger reactions than that atthe mesial side at a low magnification (Fig. 5a). At ahigh magnification of the distal side, HS was mostlylocated on the cell surface of the vascular endothelialcells and of the osteoclastic cells (Fig. 7a). In the mesialside, HS was mostly seen on the cell surface of thevascular endothelial cells, however the number of themicrovasculatures was fewer than that in the distal side(Fig. 6a). In the 2 d of Norm-15gf sample, reactivity forHS in the compressive side was more intense than inthe Norm-C sample at a low magnification (Fig. 5b). Ata high magnification, HS in the compressive side waslocated on the cell surface of the vascular endothelialcells (Fig. 6b). In the 7 d of Norm-15gf specimen, pos-itive reaction for HS was observed in the extracellularmatrix of the periodontal ligament in the compressiveside at a low magnification. The reaction for HS on thisside was more intense than that for 2 d of Norm-15gfsample (Fig. 5c). At a high magnification, HS wasobserved on the cell surface of the osteoclastic cellssurrounding the alveolar bone, and of the vascularendothelial cells. Positive reaction for HS was alsoobserved in the extracellular matrix in compressive side(Fig. 6c).

Immunoreaction for HS of the Hypo-C sampleshowed a significant decrease throughout the extra-cellular matrix in comparison with that of the Norm-Cspecimen (Fig. 6d). In the compressive side of the 2 dof Hypo-15gf sample, a weakly positive reaction wasobserved (Fig. 6e). In the 7 d of Hypo-15gf sample, amildly positive reaction for HS was observed in theextracellular matrix, especially the peripheral areawith no visible cells and fibers (Fig. 6f).

In the compressive side of the 2 d of Hypo-2gf,microvasculatures with small diameter were observedand the immunoreactivity for HS was observed on thecell surface of the endothelial cells (Fig. 6g). In the 7 dof Hypo-2gf sample, reaction for HS in the compressiveside was intense on the vascular endothelial cells andthe osteoclastic cells surrounding the alveolar bone ofthe periodontal ligament. The diameter and the numberof microvasculatures stained for HS increased from 2 dof Hypo-2gf sample (Fig. 6h). The distal side of theperiodontal ligament showed a reaction for HS on thevascular endothelial cells. The diameter of microvas-



Figure 4. Sections showing immunostaining for chondroitin sulfate (CS) at the mesial side of the distal root of mandibular right firstmolar. 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 ofHypo-2gf; h, 7 d of Hypo-2gf. CS was homogeneously observed in the extracellular matrix, with some emphasis along the fibrousstructures and around the vasculatures (a-c). In the Hypo-C sample, CS decreased significantly in comparison with Norm-C sam-ple (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 ofCS was observed around microvasculatures (arrow heads in panel h) and fibrous structure. The distribution of CS was similar to nor-mal control (h). Abbreviations: B, alveolar bone; PDL, periodontal ligament; R, root; Bar = 100 Òm.

culature in the distal side was smaller than that in thecompressive side. The number of microvasculatures inthe distal side was fewer than that in compressive side(Fig. 7b).

Discussion

The load of the Ti-Ni closed coil spring that we usedin the present study did not diminish appreciably as dis-placement of tooth increased. Therefore, we could eval-uate the response of the periodontal ligament to thecontinuous orthodontic forces with different strength.

It is found that the narrowing of microvasculaturescould not be brought about with less than 80 g/cm2 inhuman20. This pressure would be approximatelyequivalent to 10 gf in the rat maxillary first molar21.Therefore, we selected 15 gf of orthodontic force as aslightly stronger force in order not to obtain similarobservations between under normal occlusion andocclusal hypofunction. And for hypofunctional teeth, weselected 2 gf as a light force.

There are many reports on the atrophic changes inthe periodontal ligament under occlusal hypofunction1-9.

It is reported that, at 2 wks5 or 3 wks9 under hypofunc-tion, the structures of the periodontal ligament were dis-organized and the arrangement of fibers became par-allel to the alveolar walls. And at 4 wks and 12 wksunder occlusal hypofunction, the condition of the peri-odontal ligament did not appear different from that ofthe 2 or 3 wks5. Therefore, we chose to begin loadingthe orthodontic force after 3 wks post extraction of themaxillary molars. And we successfully inducedatrophic changes in the periodontal ligament.

It is generally considered that orthodontic toothmovement consists of clearly separatable three phasesoccurring in the periodontal ligament: an initial com-pressive phase consisting of changes in the visco-elas-ticity of periodontal ligament for 1-4d; a follow-up lagphase in which the tooth movement slows down for 4-7d with the appearance of the tissue with no cells andfibers in the periodontal ligament; and finally a phase inwhich the tooth moves progressively in association withbone resorption for 7-14d. It is concluded that with a 40gf in rats, the second phase was so short that the toothmovement in association with bone resorption hadoccurred by 7d11. So, we made observation on 2 d asinitial phase and 7 d as second or third phase in order


Figure 5. Sections showing immunostaining for heparan sulfate (HS) in normal group of distal root at low magni-fication (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 sidegenerally showed stronger reactions than that at the mesial side (a). Reactivity for HS in the compressive mesial sidewas more intense than in the Norm-C sample (b). In the 7 d of Norm-15gf sample, the reaction for HS was moreintense 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.


Figure 6. Sections showing immunostaining for heparan sulfate (HS) at the mesial side of the distal root of mandibular right firstmolar. 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 ofHypo-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 cellsurface of the osteoclastic cells surrounding the alveolar bone and of the vascular endothelial cells (c). In the Hypo-C sample, HSdecreased significantly in comparison with Norm-C sample (d). When tooth was applied with 15 gf, HS was observed at the periph-eral area with no cells and fibers (e,f). With 2 gf, the distribution of HS was observed on the endothelial cells (arrow heads in panelg and h) and the osteoclastic cells (arrow in panel h). The distribution of HS was similar to normal control (h). Abbreviations: B, alve-olar bone; PDL, periodontal ligament; R: root; Bar = 100 Òm.

to evaluate the distinctive remodeling for orthodonticforce.

We used CS56 antibody to detect chondroitin sulfate,and 10E4 antibody to detect heparan sulfate. CS56antibody specifically binds to Domain 2 of chondroitinsulfate22. 10E4 antibody specifically binds to N-unsub-stituted glucosamine of heparan sulfate23.

In the normal periodontal ligament, the componentsof the periodontal ligament such as the collagen fibersand the endothelial cells were arranged to resistocclusal force and mastication force. When orthodonticforce 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 oforthodontic force by immunohistochemical analysis.

In the hypofunctional periodontal ligament, thefibers became disarranged and the number of bloodvessels decreased. In the Hypo-C sample, immunos-taining for CS was weak with atrophic changes of peri-odontal ligament, similar to a previous report4.

When orthodontic force of 15 gf was applied to thehypofunctional periodontal ligament, the area with nocells and fibers was observed. In the normal periodon-tal ligament, it is reported that, the area with no cellsand fibers so-called a hyalinized tissue was observed atthe over-compressed area with 40 gf11 or 50 gf24 oforthodontic force in rat. And multi-nucleated cells

have been observed at the margin of the hyalinized tis-sue, and are speculated to resorb this tissue. Thehyalinized tissue was replaced to a new tissue. We candefine a hyalinized tissue. It is reported that chondroitin6-sulfate was present at this area and suggested thatdistribution of chondroitin 6-sulfate was related to thecompressive force in the periodontal ligament17. In thisstudy, the immunoreactivity for CS in Hypo-15gf samplewas the same result. Thus, CS may react to orthodon-tic force and participate in tissue remodeling.However, it may be that cells disappeared for somemechanism, and extracellular matrix remained. Or itmay be that hyalinized tissue have formed far from theorigin where it was synthesized. Since CS distributionof Hypo-15gf is not clear, the further study is required.

When orthodontic force of 2 gf was applied to thehypofunctional periodontal ligament, positive reactionfor CS was observed along the fibers in the 7 d sample.It is reported that mechanical stress increases collagensynthesis25. Therefore, orthodontic force of 2 gf mayhave increased the amount of collagen fibers.Meanwhile, it was reported that decorin, one of the CSproteoglycans which binds to type I collagen regulatestheir fiber formation26,27. It was also reported thatdecorin increases the diameter of collagen fibers28.Orthodontic forces might have reacted to collagenand CS synthesis and CS participate in reconstructionof collagen fibers.

HS is found to be associated with various biologicalprocesses of growth factors and cytokines, as well asbeing implicated in cell adhesion, recognition, andmigration. It is reported that the shear stress in bloodflow increases synthesis of HS in vascular endothelialcells in vitro29. And it is reported that HS may acttogether with shear stress to modify and maintain theendothelial cell configuration30. When mechanicalstress increases the expression of HS and regulatesseveral growth factors, the remodeling system of vas-cular wall may have been induced31. In the Norm-C, the2 d of Norm-15gf and the 7 d of Norm-15gf samples,there were vascular endothelial cells stained for HS ata high magnification. HS on the endothelial cell may beinterpreted to participate in vascular remodeling.

In the distal side of Norm-C and the compressiveside of 7 d of Norm-15gf sample, HS was detected onthe cell surface of the osteoclastic cells surrounding thealveolar bone. It is reported that HS binds cytokinesand protect from the decomposition and inactivation,and participated in differentiation and activation ofosteoclast32. Addition of heparin or HS inducesincreased bone resorption by osteoclasts in vitro33.


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 theendothelial cells (arrow heads in panel a) and the osteoclastic cells indistal side (arrow in panel a). The number of the microvasculatureswas more than that in the mesial side (a). A reaction for HS on thevascular endothelial cells was observed. The number of microvas-culatures in the distal side was fewer than that in mesial side. Thediameter of microvasculature in the distal side was smaller than thatin the mesial side (b). Abbreviations: B, alveolar bone; PDL, peri-odontal ligament; R, root; Bar = 100 Òm.

Under the normal condition in rat, as first molar physi-ologically moves toward the distal side, this sidebecomes compressed34,35. However, in the presentstudy, as the orthodontic force was applied to the firstmolar toward the mesial direction, mesial sidebecomes compressed. The HS positive area was situ-ated at compressed area. HS expressed on the cellsurface of osteoclastic cells may be related to the boneresorption mechanism.

When orthodontic force of 15 gf was applied to thehypofunctional periodontal ligament, HS was detectedin the peripheral area with no cells and fibers. It isreported that multi-nucleated cells have beenobserved at the margin of the tissue with no cells andfibers and are speculated to absorb the tissue inorthodontically over-compressed zones25,36. HS maytake part in the remodeling of the tissue with no cellsand fibers.

When orthodontic force of 2 gf was applied to thehypofunctional periodontal ligament, the diameter ofmicrovasculature in the 7 d sample increased in com-parison with that in the 2 d sample. It is reported that anincrease in blood flow causes the vessels dilate37. Inthe Hypo-2gf samples, orthodontic light force mayhave increased the shear stress in blood flow of peri-odontal ligament.

The present study suggested that the application ofthe orthodontic force to hypofunctional tooth is differentfrom that to normal tooth. And when forces with differ-ent strength were applied to hypofunctional periodontalligament, different reaction occurred. Distinct func-tions of CS and HS participating in tissue remodeling inresponse to the orthodontic force in the periodontal lig-ament may be performed. However, to fully understandthe functions of proteoglycan in tissue remodelingcaused by orthodontic forces, further study such asimmunohistochemistry for core proteins is required.

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