Clinical experience suggests that successfulorthodontic tooth movement can be producedwith a threshold for force duration at about 6hours, however, the changes in the periodontal lig-aments (PDL) during this period is still unclear.Nitric oxide (NO) is a marker of signal transductionrelating to bone remodeling. The aim of this studywas to observe the initial response of NO synthase(NOS) when PDL equilibrium would be brokenagainst light continuous orthodontic force. Ratmaxillary first molars were moved mesially with 2 gfTitanium-Nickel closed coil springs for 1, 3 and 6hours. The number of NADPH-diaphorase positivecells in PDL was counted for investigating NOSactivity. At the control group, NOS activity in thedistal area of the PDL was significantly higherthan that of the mesial area (P＜0.001). The activityof mesial area increased at 1-hour group (P＜0.01), while the activity of distal area droppeddown at 3- and 6-hour groups (P 3-hour＜0.05, P 6-hour＜0.001), compared with the control group.These results suggest that 1-3 hours would be thethreshold of force duration for tooth movementwith light continuous force.

Key words: orthodontic tooth movement, peri-odontal ligament equilibrium, durationof force, nitric oxide synthase

Introduction

When orthodontic force is continuously delivered to atooth, mechanical stress breaks equilibrium of peri-odontal ligaments (PDL) and the tooth will move. It isimportant to reveal how the mechanical stress istransducted to the biological response for clarifying themechanism of tooth movement. Clinical experiencesuggests that successful tooth movement can be pro-duced with a threshold for force duration at about 6hours1. An animal experiment showed that increasedlevels of cyclic adenosine monophosphate (AMP)appeared about 4 hours of sustained pressure2.However, it was the changes in the total extracts ofalveolar bone and PDL, and the changes in the PDLwhich lies on the frontline against the mechanicalstress was still unclear.

We have clarified the early response of microvascu-lature against mechanical stress and have suggestedthat the microvasculature is closely related to the signaltransduction of mechanical stress3,4. Meanwhile, nitricoxide (NO) is getting attention as a marker of vascularsignal transduction recently5,6. NO is a unique gasmediator and rapidly spreads out of cells where it isproduced and gets into neighboring cells locally7. Atfirst, NO was discovered as endothelium derivedrelaxing factor (EDRF) of the blood vessels5, however,NO is known to be produced in various cells8, and takespart in bone remodeling9, as well as regulation of the

Original Article

Duration of orthodontic force affecting initial response of nitric oxide synthase inrat periodontal ligaments

Seong-keun Yoo, Hiroyuki Warita and Kunimichi Soma

Orthodontic Science,Graduate School, Tokyo Medical and Dental University

J Med Dent Sci 2004; 51: 83–88

Corresponding Author: Hiroyuki WaritaOrthodontic Science, Graduate School, Tokyo Medical and DentalUniversity1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, JAPAN Tel/Fax: +81-3-5803-5527e-mail: warita.orts@tmd.ac.jpReceived November 7; Accepted December 19, 2003

blood vessels and nerves10. NO is synthesized bynitric oxide synthase (NOS), and NOS exists in fibrob-lasts, blood vessels and nerves of the PDL8,11. Thechanges of NO followed by experimental tooth move-ment have been reported in recent years12,13, however,initial response of NOS remains unknown when PDLequilibrium will be broken against the mechanicalstress of orthodontic force.

The aim of this study was to observe the initialresponse of NOS in the PDL against light continuousorthodontic force.

Materials and Methods

Animals and apparatus for tooth movementSixteen, 7-week-old male Sprague-Dawley rats

were used for this study. We chose male rats becauseNOS activity of female rats could be upregulated byestrogen and it would act as an undesirable factor14.Experimental group was divided into three subgroups offorce duration: 1-, 3- and 6-hour groups. Four rats wereused in each group. One hour of the force duration wasdecided on the basis of an in vitro study15, whichobserved that laminar shear stress at bovine aorticendothelial cells caused an early increase in endothe-lial NOS mRNA transcription. Six hours of the durationwas decided on the basis of clinical experiences1.Four rats without any apparatus were served as thecontrol.

The rats were anesthetized with an intraperitonealinjection of ketamin hydrochloride (40 mg/kg;Veterinary Ketalar 50�, Sankyo, Tokyo, Japan), con-taining 20% xylazine hydrochloride (4.0 mg/kg;Celactal� 2% injection, Bayer-Japan, Tokyo, Japan) asa muscle relaxant, after inhalant anesthesia withdiethylether (Wako Pure Chemical Industries, Osaka,Japan). On experiment groups, 2 gf Titanium-Nickel (Ti-Ni) closed coil spring was fixed on the bifurcation of themaxillary first molar (M1) with a hook and the gingivalthird of the upper incisors with a ligature wire, to movethe M1 mesially (Fig. 1). The both ends of the springwere reinforced with a light curing composite resin(Clearfil Liner BondⅡ, Kuraray Co. Ltd., Okayama,Japan) to prevent the spring slipping off.

All procedures were performed under the guidelinesof the Tokyo Medical and Dental University for AnimalResearch.

Fixation and tissue preparationAfter the anesthesia mentioned above, all rats were

perfused intracardially through the left ventricle with 4%paraformaldehyde and 0.2% picric acid in 0.1 M phos-phate buffer, pH 7.4. The maxillae were dissected enbloc and postfixed for 90 minutes in the same fixative,then transferred into 0.1 M phosphate buffer saline(PBS) for one night. Decalcification continued for 8weeks in 4% ethylene diamine tetraacetic acid(EDTA), pH 7.4, at 4 °C. The decalcified tissues wereimmersed in 10 and 30 % sucrose solution at 4 °C, andembedded with O.C.T. compound (Tissue-Tek� ,Sakura Finetechnical Co., Ltd., Tokyo, Japan).Fourteen Òm thick horizontal serial sections were cutwith a cryostat (Leica CM3000, Nussloch, Germany)and mounted on poly-L-lysine coated glass slides(Matsunami, Osaka, Japan).

Histochemistry of NOS activityWe chose nicotinamide adenine dinucleotide phos-

phate diaphorase (NADPH-d) enzyme histochemistryto visualize the whole activity of NOS11. The identitybetween NADPH-d and NOS activity was referred bythe past literature, with the result of copurification at bio-chemical and immunochemical assays11,16. The sec-tions were rehydrated with PBS (10 mM), then incu-bated with addition of 10 mM PBS containing 1 mM Ç-NADPH, 0.5 mM nitroblue tetrazolium, and 0.3%Triton X-100, for 3 hours in humid atmosphere at 37 °C.After the incubation, the sections were rinsed severaltimes in 10 mM PBS, air dried, and mounted with 70 %glycerin. NADPH-d positive cells were observed in thePDL of the palato-distal root of the maxillary M1. Thesections were chosen at the level of 450-550 Òm fromthe bifurcation of the root. The enzyme-stainedimages were captured with a digital camera(DXm1200, Nikon, Tokyo, Japan) mounted on a lightmicroscope (Nikon Microphoto-FXA, Nikon, Tokyo,Japan), and were stored in a 24 bits true color TIFF for-

S. YOO, H. WARITA and K. SOMA J Med Dent Sci84

Fig. 1. A schematic drawing of the experimental modelC: 2gf Titanium-Nickel closed coil spring, M1: maxillary first molar, H:hook, I: maxillary incisors, L: ligature wire

mat.

Quantitative AnalysisThe number of NADPH-d positive cells was counted

in the mesial and distal area of the PDL of palato-distalroots for detecting NOS activity. The measurementareas were defined with the alveolar bone surface, theroot surface, and two tangential lines parallel to the linewhich connected the centers of palato-mesial andpalato-distal roots (Fig. 2). Each group had 4 differentsections for analysis. Two investigators counted thenumber of NADPH-d positive cells in the PDL byusing a single-blind method with an image analysissoftware (Image-Pro, Media Cybernetics, SilverSpring, MD, USA).

The number of NADPH-d positive cells was analyzedby ANOVA, followed by Scheffe’s post hoc test (P＜0.05), using Statview 5.0J (SAS Institute, Cary, NC).

Results

Control groupThe distal area of PDL was narrower than the

mesial area (Fig. 3). NADPH-d activity was observed atthe blood vessels, fibroblastic cells, and osteoclast-likecells in the PDL. The number of NADPH-d positive cellsin the distal area was significantly higher than that ofthe mesial area (p＜0.001, Fig. 4).

1-hour groupThe mesial and distal width of PDL was reversed at 1

hour, that is, the mesial area became narrower than thedistal area (Fig. 3). The blood vessels, fibroblastic cells,

and osteoclast-like cells were mainly stained withNADPH-d. NADPH-d activity in the mesial areaincreased significantly compared with the mesial areaof the control (p＜0.01) and it was almost equivalent tothe distal area of the control (Fig. 4). The activity in thedistal area of 1-hour group increased in some degree,however, it was not significantly higher than that in thedistal area of the control. There was no significant dif-ference of the NADPH-d activity between the mesialand the distal area of 1-hour group.

3-hour groupThe mesial width of PDL in 3-hour group was much

narrower than that of 1-hour group (Fig. 3). NADPH-dactivity was observed at the blood vessels, fibroblasticcells, and osteoclast-like cells in the PDL. At themesial area, the number of NADPH-d positive cells in3-hour group decreased compared with that in 1-hourgroup, and declined to the level of the control (Fig. 4).At the distal area, the activity in 3-hour groupdecreased compared with the distal area of the controland 1-hour group, and declined to the level of themesial area of the control (Fig. 4). Additionally, therewas no significant difference of the NADPH-d activitybetween the mesial and the distal area.

6-hour groupThe width of PDL and the distribution of NADPH-d

positive cells in 6-hour group were similar to those ofthe 3-hour group. The NADPH-d activity in 6-hourgroup showed the same tendency with the 3-hourgroup.

Discussion

The NOS activity in the controlThe distal area in PDL is pressure side because rat

molars move distally in the physiological condition17. Asa result, shear stress in the blood vessels would rise atthe distal area18. Increase in number of NADPH-dpositive cells in the distal area shows accordance within vitro studies which observed the increase of shearstress in a blood vessel raised the NO production15,19.NO is hard to be detected because it is a gas mediatorand NOS consists of some isoforms. Therefore, westained the whole NOS with NADPH-diaphorase toobserve the total activity of NOS.

The changes in the mesial areaThe mesial area was tension side at the control, how-

85DURATION OF FORCE AFFECTING THE RESPONSE OF NOS IN RAT PDL

Fig. 2. A scheme of the measurement areasPM: palato-mesial root, PD: palato-distal root, PDL: periodontal liga-ment, AB: alveolar bone, M: mesial area of the measurement, D: dis-tal area of the measurement

ever, it turned into a pressure side with the applicationof mesially directional orthodontic force. At 1-hourgroup, NOS activity rose up to the same level of the dis-

tal area of the control, namely, the physiological pres-sure side. This change was thought to be the increaseof shear stress, similar to the pressure side of the con-

S. YOO, H. WARITA and K. SOMA J Med Dent Sci86

Fig. 3. Palato-distal roots of the maxillary first molars with NADPH-diaphorase stainingNOS activity was observed at blood vessels, fibroblastic cells, and osteoclast-like cells (arrow heads) . The distal area was narrower than the mesial area in the control group (Cont.). The mesial area became narrower than the distal area in 1-hourgroup (1h). The mesial width of PDL in 3-hour group (3h) was much narrower compared with that of 1-hour group. The width and the distrib-ution of NADPH-diaphorase positive cells in 6-hour group (6h) were similar to those of 3-hour group.M : mesial, D : distalFB : fibroblasts, BV : blood vessels, OC : osteoclast-like cells�: direction of physiological tooth movement�: direction of experimental tooth movement Scale bar represents 200 ÒmThe images in the box were magnified 200 times.

trol. This results coincides with an in vitro experi-ment15 in which the NOS activity increased after the 1hour of mechanical stress. Furthermore, the reasonwhy the NOS activity decrease in 3- and 6-hour groupmight be as follows; we used light continuous force tomove teeth, however, the experimental condition was arapid change compared with the physiological state,and the excessive production of NO could be cytotoxicfor the cell function20.

The changes in the distal areaThe distal area was a pressure side at the control,

however, it turned into a tension side with the applica-tion of the mesially directional orthodontic force.Unlike the experimental mesial area, the distal area in1-hour group showed no significant difference. At 3hours, NOS activity decreased to the level of themesial area of the control, which was the physiologicaltension side. It was the same tendency in the 6-hourgroup. The results were supported by an in vitroreport which found that the eNOS mRNA half-life was 5hours in unshear stressed endothelial cells15.

The role of NO in the signal transmission andbone remodeling

On our experiment, the number of NADPH-d positivecells changed in 1-3 hours. This means NOS activity

changed in 1-3 hours, accordingly, the production of NOwould be changed. NO has been reported to increasethe microvascular permeability21, and it might have therelation with the role of NO at initial stage of boneremodeling, because monocytes in the blood vesselsbecome the basis of bone remodeling7.

To summarize the changes in the mesial and the dis-tal area of PDL, light continuous orthodontic forcechanged the NOS activity in the PDL at 1-3 hours. Theresults suggested that the PDL equilibrium could bebroken at shorter duration of light continuous ortho-dontic force than that formerly reported1,2.

Acknowledgements

This study was financially supported by Grants-in-Aidfor Scientific Research (14370688, 14571941) from theMinistry of Education, Culture, Sports, Science andTechnology, Japan. Part of this study was presented atthe 61st annual meeting of the Japanese OrthodonticSociety, Nagoya, Japan, October 22-24, 2002.

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