Biomechanical and biochemical factors areinvolved in bone remodeling. Occlusal loading is awell-known mechanical modulator of alveolarbone remodeling. Neuropeptides, such as vasoac-tive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), have been describedwithin the biochemical bone regulators. In thisstudy, the influences of occlusal loading on thealveolar bone remodeling and the distribution ofVIP- and CGRP-immunoreactive (IR) fibers wereinvestigated 1, 3 and 5 days after tooth replantationin rats. At day 5, occlusal loading induced a signif-icant increase (p<0.05) in osteoclast number andosteoblast surface compared to those in the non-occluded group. VIP-IR fibers were observedbeside osteoblastic layers and their distributionwas significantly enhanced (p<0.05) at day 5 in theoccluded group, compared to the non-occludedgroup. Although there was immunoreactivity forCGRP in the periodontal ligament and alveolarbone apically, CGRP-IR fibers were not detectedabove the furcation. These results suggest that,after tooth replantation in rats, occlusal loading

induced an increase in osteoclast and osteoblastformation, and that VIP might play a functional rolein osteoblasts.

Key words: occlusal loading, neuropeptides, VIP,CGRP, alveolar bone.

Introduction

Bone tissue is continuously remodeled through theactivities of bone-forming osteoblasts and bone-resorbing osteoclasts.1 It is well known that mechanicalloading plays a role in bone remodeling, however, themechanism(s) by which loading stimuli on bone aretranslated into biomechanical stimuli that regulatebone remodeling are not fully understood.2

Bone cells are considered to respond directly or indi-rectly to local strains in their vicinity due to loading innormal function. These strains are the product of thebones’ external loads and their structural properties andthus include the information necessary to be the con-trolling input for adaptive bone modeling and remodel-ing.3,4

The regulation of bone remodeling is a complexprocess that involves the activities of and interactionsbetween different bone cells that are regulated by avariety of systemic hormones, cytokines, growth factorsand inflammatory mediators. Another proposed regu-latory element is the nervous system, which, through

Original Article

Effects of occlusal loading on alveolar bone remodeling and changes in the distri-bution of neuropeptides after tooth replantation in rats

Isis Barros1, Takeshi Muramoto2 and Kunimichi Soma3

1) Graduate student, Orthodontic Science, Department of Orofacial Development and Function, Division ofOral Health Science, Graduate School, Tokyo Medical and Dental University (corresponding author)2) Orthodontic Science, Department of Orofacial Development and Function, Division of Oral HealthScience, Graduate School, Tokyo Medical and Dental University3) Professor and Chairman, Orthodontic Science, Department of Orofacial Development and Function,Division of Oral Health Science, Graduate School, Tokyo Medical and Dental University

J Med Dent Sci 2007; 54: 1–149

Corresponding Author: Isis BarrosOrthodontic Science, Department of Orofacial Development andFunction, Division of Oral Health Science, Graduate School, TokyoMedical and Dental University,1-5-45 Yushima, Bunkyo-ku, Tokyo113-8549, JapanE-mail address: isisorts@yahoo.co.jp (corresponding author)Tel/fax: +81-3-5803-5528Received October 12; Accepted December 1, 2006

the release of neuronal messengers, has been sug-gested to participate in bone metabolism.5,6

Vasoactive intestinal peptide (VIP) and calcitoningene-related peptide (CGRP) are the two neuropep-tides which have been most extensively studied in thisfield. These neuropeptides affect the activities ofosteoblasts as well as osteoclasts.7-10 Moreover,immunoreactive (IR) fibers for VIP and CGRP havebeen demonstrated on the periodontal ligament (PDL)and alveolar bone (AB).11-13 Changes in the distributionof these fibers have been described after experimentaltooth movement.13,14

Compared to other bone tissues in the body, the ABis subjected to continual and rapid remodeling associ-ated with the functional demands of mastication.15 Ithas been suggested that the PDL plays a role in themechanotransduction of signals required for bonehomeostasis and participates in the regulation ofalveolar bone volume.16

Tooth transplantation is a valuable treatment optionfor replacing missing or extracted teeth and has beenlargely used in conjunction with orthodontic treat-ment.17,18 While previous studies have focused on theeffects of occlusal force on the healing of the PDL aftertooth replantation,19,20 little is known about the effects ofocclusal force on AB remodeling after this procedure.Since tooth replantation causes rupture of the PDL andimpairs its mechanical properties, changes in ABremodeling are expected. Therefore, the purposes ofthis study were to investigate, the effects of occlusalloading on AB remodeling and changes in the disposi-tion and distribution of VIP- and CGRP-IR fibers on theAB after molar replantation in the rat.

Materials and Methods

Animals and experimental modelTwenty-four male 5-week-old Sprague-Dawley rats

(Sankyo Lab Service Corporation, Inc., Tokyo,Japan), body weight 144± 7g (mean± SD), wereused in this study. The animals were selected due tothe pulpal and periodontal characteristics of rat at thisage. To address the influence of occlusal loading onbone remodeling after tooth replantation, the experi-mental group was divided into occluded (n = 12) andnon-occluded groups (n = 12).

All the procedures were performed under anesthesia.After the administration of diethyl ether for anesthesia,the animals were deeply anesthetized by theintraperitoneal injection of chloral hydrate (400

mg/Kg). The right maxillary first molars of the animalswere replanted according to the method described byKvinnsland.21 The molars were loosened, the rootswere rotated anteriorly so that they came out of thesocket while leaving part of the attached mesial gingivaintact, and then immediately repositioned. No postop-erative splinting was used. Postoperative antibiotictreatment consisted of amoxicillin (32.4 mg/Kg). Thecontralateral first molars of the occluded group animalsserved as control; these specimens were denominatedas non-replantation group.

According to the method developed by Suhr,22 in thenon-occluded group an anterior bite-plate and a metalcap constructed from band material (4.6 x 0.13 mm;Tomy International, Tokyo, Japan) were attached to themaxillary and mandibular incisors, respectively, usinglight-curing composite resin (Clearfil Photo SC,Kuraray Inc., Okayama, Japan). The appliance wasdesigned to impede occlusal function in the molarregion, whereas in the occluded group, occlusal contactwas maintained.

Rats in the non-occluded group were fed liquid diet(Liquid fodder, Clea Japan Inc, Shizuoka, Japan) andthe ones in the occluded group were fed standard ratchow (CE-2, Clea Japan Inc, Shizuoka, Japan), both adlibitum. Both groups had free access to drinkingwater. The animals on both groups were sacrificed at 1,3 and 5 days after tooth replantation. All procedures fol-lowed the guidelines of the Tokyo Medical and DentalUniversity for Animal Research and were approved bythe Animal Ethics Committee.

Tissue preparationAfter deep anesthesia, the animals were sacrificed by

transcardiac perfusion of 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The upper jaws wereexcised en bloc and further immersed in the same fix-ative solution at 4°C for 12 hours. After being rinsedwith PBS, the specimens were decalcified in 4.13%EDTA-2Na (ethylene diamine tetra acetic acid disodi-um) solution at 4°C for 4-5 weeks and embedded inparaffin by conventional methods. Five-Òm-thick serialsagittal sections were cut parallel to a line that passedthrough the disto-buccal and mesial roots of the firstmaxillar molar (M1) (RM 2155, Leica Co LTDA,Nussloch, Germany); these sections also included thesurrounding tissue (Figs. 1A and 1B).

ImmunohistochemistryTo evaluate the morphology of the PDL and AB, and

the time-related changes throughout the experimental

I. BARROS, T. MURAMOTO and K. SOMA J Med Dent Sci2

period, some sections were stained for hematoxylin andeosin (HE) by conventional methods.

To analyze the disposition and distribution of neu-ropeptidergic fibers and bone cells at the AB, double-staining was carried out. Double-staining was per-formed to reveal immunoreactivity for neuropeptidesand tartrate-resistant acid phosphatase (TRAP) in thesame sections. Serial sections were stained for eitherVIP or CGRP while alternating the first antibody.Immunohistochemical studies were performed onparaffin-embedded sections using polyclonal antibodyto VIP diluted 1:8000 (Euro-Diagnostica, Malmo,Sweden) and to CGRP diluted 1:2000 (YanaiharaInstitute, Shizuoka, Japan). First-antibody reactivitywas revealed by Catalyzed Signal Amplification (CSA)System HRP (Dako, Carpinteria, CA, USA). The pro-tocol followed was essentially that described byTolcos.23 After color development, TRAP staining wasperformed on the same sections. The sections wereincubated in de-ionized water containing naphthol AS-MX phosphate (Sigma, St. Louis, MO, USA) as the sub-strate and Fast Red Violet LB salt (Sigma, St. Louis,MO, USA) for a color reaction at pH 5.4 with 50 mMsodium tartrate. The color reaction was carried out in ahumid chamber under 37°C for 15-20 minutes and thereaction was then stopped by washing with distilledwater. The sections were counterstained with hema-toxylin, mounted with aqueous mounting medium(Gel/ Mount, Biomeda Corp., Foster City, CA, USA) andcover-slipped. Negative controls were prepared byreplacing the primary antibody with TRIS bufferedsaline-Tween (TBST) buffer (0.05 M Tris-HCl [pH 7.6],0.3 M NaCl, and 0.1% Tween 20). In these negativecontrols, immunoreactivity was not detected (data notshown). The sections were observed and pho-tographed by a light microscope (Nikon Microphoto-FXA, Nikon, Tokyo, Japan) equipped with a digital cam-

era (DXm1200, Nikon, Tokyo, Japan).

HistomorphometryHistological examination focused on the interradicu-

lar area. Since there were signs of distal drift in all ofthe groups throughout the experimental period, and themost remarkable changes occurred at the interradicularbone adjacent to the furcation, the histomorphometricparameters were measured in a quadrangular area(400x400) µm2 on the AB alongside the disto-buccalroot (Fig. 1A).

The measurements and calculations for bone histo-morphometry were referenced to the standardnomenclature described by Partiff.24 The parametersmeasured included osteoclast number (N.Oc/BS,#/mm) and osteoblast surface (Ob.S/BS, %).Osteoclasts were identified as multinucleated TRAP-positive (red-staining) cells that were situated at thebone surface. Osteoblasts were identified as cuboidalcells that lined the bone surfaces. The measurementswere performed as described by Yamashiro.25

Since CGRP-IR fibers were not regularly found onthe evaluated area, measurements were only per-formed for the area of VIP-IR fibers.

Four rats were evaluated in each group. The mea-surements were made with image analysis software(Image-Pro, Media Cybernetics, Silver Spring, MD,USA). The data were analyzed by one-way analysis ofvariance (ANOVA) or Kruskal-Walli test for intergroupcomparisons. To detect significant changes over timebetween each experimental group and the non-replantation group, and between the experimentalgroups, the data were compared using repeated mea-sures ANOVA with Bonferroni post-hoc-test.Statistical analyses described above were done usingSPSS version 10.0J (Chicago, IL, USA). Data areexpressed as mean± SD. The level of significancewas set at 0.05.

Results

The body weight in the experimental animals regu-larly increased during the experimental period. Therewas no significant difference in the mean body weightbetween the groups (data not shown), which indicatesthat differences in diet or dietary consistency had noeffect on the animals’ general growth weight anddevelopment.

3OCCLUSAL LOADING & NEUROPEPTIDES ON BONE REMODELING

Fig. 1. (A) Schematic drawing of a sagittal section of M1. The mea-surements for bone histomorphometry were made within a quadran-gular area (400x400) µm2 on the AB adjacent to the furcation. (B)Diagram showing a horizontal view of M1. The sections were cut par-allel to the indicated line. M1, upper first molar; M, mesial; D, distal;B, buccal; L, lingual.

Histological ObservationAt day 1, in the line of PDL rupture, blood cells were

observed and a lack of continuity between the alveolarand root portions of the PDL was evident. Also, disrup-tion of the normal blood supply occurred (Figs. 2A and2D). At day 3, the first signs of re-arrangement of PDLcells were observed with concomitant tissue revascu-larization. Continuity between the alveolar and root por-tions of the PDL was re-established in both groups.Numerous osteoclasts were observed on the AB in theoccluded group, but were scarce in the non-occludedgroup (Figs. 2B, 2E, 3B and 3E). At day 5, PDL healinghad progressed, although disorientation of the peri-odontal fibroblasts was still evident. In the occludedgroup, the bone surface was covered by numerousosteoblasts, and in the non-occluded group a large areaof the bone surface was covered by hard tissue (Figs.2C, 2F, 3C and 3F). At days 3 and 5, whereas the PDLin the occluded group animals was apparently widerthan that in the non-replantation group ones, in the non-

occluded group the PDL thickness appeared to be sim-ilar to that in the non-replantation group animals(Figs. 2B, 2C, 2E, 2F and 2G).

VIP- and CGRP-IR fibers were usually found alongblood vessels, however isolated fibers were also iden-tified. VIP-IR fibers were often found bordering areasthat were covered by osteoblasts (Fig. 3). At day 1,although there was no reactivity for VIP-IR fibers,such activity was observed in both groups at days 3and 5 inside the AB (Fig. 3). Although CGRP-IR fiberswere observed in the PDL apically and in superior por-tions of the AB and bone marrow, in the study areathese fibers were not detected (Fig. 4).

HistomorphometryThe data regarding osteoclast number, osteoblast

surface and VIP-IR fiber area are expressed graphicallyin Fig. 5.

Osteoclast number and osteoblast surface At days 1, 3 and 5, osteoclast number in the

I. BARROS, T. MURAMOTO and K. SOMA J Med Dent Sci4

Fig. 2. HE sections of the AB-PDL complex adjacent to the furcation. (A), (B), (C),occluded group days 1, 3 and 5, respectively; (D), (E), (F), non-occluded group days 1,3 and 5, respectively; (G) non-replantation group. (A) and (D) At day 1, disruption of thePDL (asterisks) was caused by the replantation procedure. (B) and (E) At day 3, con-tinuity of the PDL was restored which coincided with tissue revascularization.Numerous osteoclasts were observed on the bone surface in the occluded group(arrows). (C) and (F) Several osteoblasts (ob) covered the bone surface in the occlud-ed group, whereas hard tissue was common in the non-occluded group. AB, alveolarbone; PDL, periodontal ligament. Scale bar = 200 Òm.

5OCCLUSAL LOADING & NEUROPEPTIDES ON BONE REMODELING

Fig. 3. Reactivity for VIP-IR fibers (brown) and TRAP (red) in thealveolar bone in the occluded group days 1, 3 and 5, (A), (B) and (C),respectively and in the non-occluded group days 1, 3 and 5, (D), (E)and (F), respectively. (C’) and (F’) are insets from (C) and (F). (A) and(D) At day 1 immunoreactivity for VIP was not observed. (B) and (E)Numerous osteoclasts (arrows) covered the bone surface in theoccluded group. In both groups, VIP-IR fibers (arrow heads) wereidentified along blood vessels usually bordering areas covered byosteoblasts. (C) Intense VIP immunoreactivity was observed in theoccluded group. (C’) and (F’) Notice the close association betweenVIP-IR fibers and osteoblastic layers. ob, osteoblast; v, blood vessel.Scale bar = 100 Òm.

Fig. 4. Immunoreactivity for VIP (A) and CGRP (B) on serial sec-tions. Throughout the experimental period VIP-IR fibers (arrowheads) were observed along blood vessels, whereas immunoreac-tivity for CGRP was not detected (asterisk) in the alveolar bone abovethe furcation. v, blood vessel. Scale bar = 100 Òm.

Fig. 5. Histomorphometric analyses of osteoclast number, osteoblastsurface and VIP-IR fiber area, in the alveolar bone, in occluded andnon-occluded groups. Each value is the mean ± SD (n = 4). (a)Significant difference (p<0.05) between the occluded group and thenon-replantation group. (b) Significant difference (p<0.05) betweenthe non-occluded group and the non-replantation group. (*)Significant difference (p<0.05) between the occluded and non-occluded groups.

occluded group was significantly increased comparedto that in the non-replantation group (p<0.05).However, in the non-occluded group, osteoclast numberwas significantly increased compared to that in the non-replantation group only at day 1 (p<0.05). At days 3 and5, osteoclast number in the occluded group was signif-icantly increased compared to that in the non-occludedgroup (p<0.05).

At days 3 and 5, osteoblast surface in the occludedgroup was significantly increased compared to that inthe non-replantation group (p<0.05). However,osteoblast surface in the non-occluded group was sig-nificantly increased compared to that in the non-replantation group only at day 3 (p<0.05). At day 5,compared to the non-occluded group, there was a 2-fold increase (p<0.05) in osteoblast surface in theoccluded group.

VIP-IR fiber areaSince immunoreactivity for VIP-IR fibers was not

observed at day 1 in the AB on both experimentalgroups, the results presented reflect days 3 and 5. Atday 3, there were neither significant differencesbetween the experimental groups, nor between eachexperimental group and the non-replantation groupregarding VIP-IR fiber area. However, compared to thenon-replantation group, there was a 2-fold increase(p<0.05) in VIP-IR fiber area in the occluded group atday 5. Significant differences between the experimentalgroups were seen at day 5; compared to the non-occluded group, there was a 2-fold increase (p<0.05) inVIP-IR fiber area in the occluded group.

Discussion

The results of this study demonstrated that toothreplantation and its association to occlusal loadinginduced significant changes in alveolar bone remodel-ing. From the results it can be suggested that toothreplantation induced the increase in osteoclast numberin both experimental groups at day 1. It has beenreported that the initial reaction to the injury caused bytooth transplantation is always acute inflammation.26 Inaddition, it is known that inflammatory mediators alsoplay a role in the formation of osteoclasts.1 Takentogether, it can be surmised that the inflammationcaused by the replantation process might have partici-pated in the increase in osteoclastogenic events in bothexperimental groups at day 1.

Taken the results, it can be also suggested thatocclusal loading induced enhance in osteoclastogenic

events at days 3 and 5, and enhance in osteoblast for-mation at day 5. Therefore, occlusal loading signifi-cantly increased alveolar bone remodeling above thefurcation after tooth replantation. The effect ofocclusal loading in osteoclastogenic events in thisstudy is in agreement with previous studies whichhave demonstrated that compressive forces induceosteoclast activity.13,27 In this study, the fact that therewas no significant difference in osteoclast numberbetween the experimental groups at day 1, may be dueto the fact that the replantation procedure damaged thePDL with a disruption of PDL continuity. The firstsigns of PDL re-arrangement on day 3 coincided withan increase in osteoclast number in the occludedgroup, indicating that forces due to occlusal loadingwere transduced to the bone, thus enhancing osteo-clastogenic events. Even though the mechanicalproperties of the PDL were reduced after toothreplantation,28 it can be assumed that the forces fromocclusal loading were transduced to the bone andevoked an increase in osteoclastogenic events in theoccluded group. It is important to emphasize that inbone, osteocytes and osteoblasts are considered toexhibit mechano-sensation properties3,4 and thereforemight have also become sensitized to mechanicalstimuli by occlusal loading.

In addition, it can be suggested that an increase inosteoblast formation was induced by tooth replantationin both experimental groups at day 3, and by occlusalloading in the occluded group at day 5. In both circum-stances, the increase in osteoblast formationoccurred following an increase in osteoclast formation.It has been reported that in bone remodeling,osteoblastic bone formation occurs in a programmedprecise and quantitative manner following osteoclasticbone resorption.1 Therefore, it can be surmised that theincrease in the number of active osteoclasts might havecaused enhance in bone resorption and might have atleast partially induced enhance in osteoblast formationin order to repair the resorbed bone surface.

It has been reported that traumatic occlusion of thegraft after tooth transplantation is significantly related toroot resorption activity.29 In this study, occlusal loadingwas also significantly related to the enhance in alveolarbone remodeling after tooth replantation. Taken theeffects of mechanical loading on root resorption activi-ty and on bone remodeling, it would be advisable toavoid excessive loading of the grafts on early stagesafter tooth transplantation. In contrary, it is known thatmasticatory stimuli reduces ankylosis after experi-mental tooth replantation,30 therefore, further studies on

I. BARROS, T. MURAMOTO and K. SOMA J Med Dent Sci6

the amount of forces and the proper time of force appli-cation are necessary to achieve optimal results aftertooth transplantation.

The influence of VIP on bone tissue has been thesubject of several studies. VIP-IR fibers, which are ofsympathetic origin at the head, have been observed inareas with high osteogenic activity.5,31 In addition to thefact that chemical-induced sympathectomy resulted inthe loss of nerve fibers containing VIP with a concomi-tant 45.5% increase in osteoclast number,32 boneapposition and the mineralization rate at the mandiblewere significantly lower following surgical sympathec-tomy. These changes were concurrent with a significantincrease in the number of osteoclasts per bone surfaceof the molar root socket.33 VIP-specific binding sites andVIP receptors have been described on osteoclasts andosteoblasts.34-36 Furthermore, it has been suggestedthat VIP contributes to the formation of committedosteoblasts and the regulation of osteoblastic differen-tiation.6 VIP has also been reported to stimulate boneformation in vitro, by up-regulating alkaline phos-phatase activity in osteoblasts.8 Moreover, VIP inhibitedthe motility of differentiated osteoclasts and osteoclastformation, probably by regulating the expression ofRANK, RANKL and OPG.7 In this study, the fact thatVIP-IR fibers were preferentially distributed borderingosteoblastic layers and the increase in the occludedgroup on day 5, which coincided with an increase inosteoblast surface, might indicate that VIP plays a func-tional role in the regulation of osteoblast formation.

An anabolic effect of CGRP on bone remodelingthrough a decrease in bone resorption and the stimu-lation of bone formation has been described in vitro andin vivo,9,10,37,38 whereas the participation of CGRP-IRfibers in osteoclastogenesis after experimental toothmovement has been described in dental tissue inrats.13 In this study, CGRP-IR fibers were not found onthe AB above the furcation and there were nochanges in their distribution throughout the experi-mental period, indicating that CGRP might not partici-pate in bone-regulatory events in this area soon aftertooth replantation.

Although bone remodeling is a complex process,which involves the activities of numerous mediators, thepresent results suggest that after tooth replantation,occlusal loading induced an increase in osteoclast andosteoblast formation, and that VIP might play a func-tional role in osteoblast formation.

Acknowledgments

This research was supported by Grants-in-Aid forScientific Research (nos. 15592158 and 15791202)from the Ministry of Education, Culture, Sports,Science and Technology, Japan.

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