tooth movement

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DOI: 10.1177/10454411910020040101 1991 2: 411CROBM

Zeev DavidovitchTooth Movement

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Critical Reviews in Oral Biology and Medicine, 2(4):411-450 (1991)

Tooth MovementZeev DavidovitchDepartment of Orthodontics, The Ohio State University College of Dentistry, Columbus, Ohio

ABSTRACT: This article reviews the evolution of concepts regarding the biological foundation of force-inducedtooth movement. Nineteenth century hypotheses proposed two mechanisms: application of pressure and tensionto the periodontal ligament (PDL), and bending of the alveolar bone. Histologic investigations in the early andmiddle years of the 20th century revealed that both phenomena actually occur concomitantly, and that cells, aswell as extracellular components of the PDL and alveolar bone, participate in the response to applied mechanicalforces, which ultimately results in remodeling activities.

Experiments with isolated cells in culture demonstrated that shape distortion might lead to cellular activation,either by opening plasma membrane ion channels, or by crystallizing cytoskeletal filaments. Mechanical distortionof collagenous matrices, mineralized or non-mineralized, may, on the other hand, evoke the development ofbioelectric phenomena (stress-generated potentials and streaming potentials) that are capable of stimulating cellsby altering the electric charge on their membrane or their fluid envelope. In intact animals, mechanical pertur-bations on the order of about 1 min/d are apparently sufficient to cause profound osteogenic responses, perhapsdue to matrix proteoglycan-related "strain memory".

Enzymatically isolated human PDL cells respond biochemically to mechanical and chemical signals. Thelatter include endocrines, autocrines, and paracrines. Histochemical and immunohistochemical studies showedthat during the early places of tooth movement, PDL fluids are shifted, and cells and matrix are distorted.Vasoactive neurotransmitters are released from periodontal nerve terminals, causing leukocytes to migrate outof adjacent capillaries. Cytokines and growth factors are secreted by these cells, stimulating PDL cells andalveolar bone lining cells to remodel their related matrices. This remodeling activity facilitates movement ofteeth into areas in which bone had been resorbed.

This emerging information suggests that in the living mammal, many cell types are involved in the biologicalresponse to applied mechanical stress to teeth, and thereby to bone. Essentially, cells of the nervous, immune,and endocrine systems become involved in the activation and response of PDL and alveolar bone cells to appliedstresses. This fact implies that research in the area of the biological response to force application to teeth shouldbe sufficiently broad to include explorations of possible associations between physical, cellular, and molecularphenomena. The goals of this investigative field should continue to expound on fundamental principles, partic-ularly on extrapolating new findings to the clinical environment, where millions of patients are subjected annuallyto applications of mechanical forces to their teeth for long periods of time in an effort to improve their positionin the oral cavity. Recently developed research tools such as cell culture techniques and immunologic probes,are the best hope for enhancing this development.KEY WORDS: orthodontic forces, distortion of cells and matrix, neurotransmitters, cytokines, synergism.

I. INTRODUCTORY REMARKS

Throughout their natural history, teeth moveand migrate. Prior to their eruption into the oralcavity, changes in the position of tooth buds oc-cur primarily due to the growth of dental struc-tures, and the concomitant remodeling of neigh-boring tissues, i.e., alveolar bone, gingiva, andperiodontal ligament (PDL), including the dentalfollicle. Following their emergence into the oralcavity, teeth reach a position in the dental arch,

dictated by the forces of the surrounding musclesof the tongue, cheeks, and lips, and by contactwith teeth of the opposite jaw. During mastica-tion, teeth can move slightly in the vertical andhorizontal directions, within the constraints of thesoft tissues of the PDL, and the bendability ofthe alveolar bone. Despite their large magnitude,masticatory forces do not alter the position ofteeth, due to their short duration. However, inthe presence of periodontal disease, when para-dental tissues are gradually destroyed, teeth can

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migrate into new positions, where the mastica-tory and parafunctional forces reach equilibrium.Often, these new positions are aestheticallyundesirable.

Tooth position may be deemed undesirabledue to functional and aesthetic considerations,prompting patients to seek orthodontic care. Inits most simplistic translation, "orthodontics"means straightening teeth. This "straightening",or movement of teeth into desirable positions, isaccomplished by the application of forces to teeth,usually of small magnitude (on the order of a fewgrams per square centimeter of dental root sur-face) and long duration (usually about 2 years).Millions of people are subjected annually to or-thodontic treatment worldwide, making thisbranch of dental care a widespread and lucrativespecialty. The number of dentists in the U.S. wholimit their practice to orthondontics is presentlyaround 10,000. However, many general dentistsworldwide provide orthodontic care to their pa-tients, and all, specialists and generalists alike,base their therapeutic means upon the time-testedobservation that teeth can be forced to move awayfrom their position in the dental arch to new lo-cations by means of applied mechanical forces.

The following review focuses upon the phe-nomenon of tooth movement that can be broughtabout by the application of continuous mechan-ical forces to teeth. Excluded from this revieware the phenomena of eruptive, pathological (per-iodontal disease related), and surgically inducedtooth movements. Specifically, this review dis-cusses biological aspects of force-induced toothmovement on the tissue, cellular, and molecularlevels.

II. HISTORICAL PATHFINDERS

The first recorded recommendation to useforce for orthodontic reasons was made aroundthe year 1 A.D. by Celsus, who suggested theapplication of finger pressure to teeth for align-ment purposes.1 Seventeen centuries later, Fau-chard was the first to publish a description andan illustration of an orthodontic appliance, whichgenerated forces by using ligatures to tie teeth toa rigid arch.2 In the 18th century, Hunter3 pro-vided the first biological explanation for ortho-

dontic tooth movement: "To extract an irregulartooth would answer but little purpose, if no al-terations could be made in the situation of therest; but we find that the very principle uponwhich teeth are made to grow irregularly is ca-pable, if properly directed, of bringing them evenagain. This principle is the power which manyparts (especially bones) have of moving out ofthe way of mechanical pressure."

Two significant observations were made dur-ing the 19th century concerning the biologicalnature of orthodontic tooth movement. In 1815,Delabbare4 remarked that pain and swelling ofparadental tissues occur following the applicationof orthodontic forces to teeth. In contemporaryterms, Delabbare introduced the notion that in-flammation is an integral part of orthodontic toothmovement. In 1888, Farrar5 hypothesized thattooth movement is due, at least in part, to bendingof alveolar bone by applied forces. This notionwas supported by Wolffss6 proposition in 1892that the internal architecture of bone is dictatedby the mechanical forces that act upon it.

The first report on the histomorphology oftissues surrounding orthodontically treated teethwas published by Sandstedt in 1904 to 1905.7T8That landmark experiment, which was performedin one dog, concluded that force-induced tissuechanges are limited to the PDL and its alveolarbone margin. At the end of 3 weeks of treatment,Sandstedt observed new bone growth in thestretched PDL, and bone resorption in the areaof PDL compression. Cell death occurred in thecompressed PDL when the applied force was ex-cessive, and the alveolar bone resorbed as a resultof osteoclastic activity in adjacent marrow spaces(undermining resorption).

Six years later, Oppenheim9 reported on ahistologic examination of thejaws of one juvenilebaboon whose teeth had been treated by ortho-dontic forces for 40 d. In contrast to Sandstedt,Oppenheim saw no demarcation between the oldand new alveolar bone near the moving teeth,but rather a trabecular structure that strongly sug-gested a complete transformation of the entirealveolar bone in that region. The bony trabeculaewere all rearranged in the direction of the force.However, Oppenheim's conclusions that ortho-dontic forces were capable of transforming theentire alveolus were rejected by his contempor-

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aries as misinterpretations. He was also criticizedfor using an animal with deciduous teeth for hisexperiment, suggesting that the transformation hehad seen was related to growth and developmentrather than being the outcome of applied me-chanical forces.

Oppenheim's "transformation" hypothesismight have supported Farrar's earlier contentionthat orthodontic forces bend the alveolar bone,and thus are able to stimulate all the cells in andaround this bone. However, Farrar's clinical ap-proach also was not popular, because he advo-cated the use of heavy forces that could indeedbend the bone. In fact, the pendulum swung fur-thest to the other side, when Schwarz10 recom-mended the use of light orthodontic forces. Hedefined those forces as being "not greater thanthe pressure in the blood capillaries" (15 to 20mmHg, or about 20 to 26 g/cm2 of root surface).

A 1957 publication by Fukada and Yasuda"lattracted wide attention. They observed thatbending of bone by mechanical means evokes thegeneration of measurable electric potential spikesin areas of compression and tension. While thisobservation caused the rebirth of the field of ap-plied exogenous electricity to bone nonunionfractures, it also precipitated the reintroductionof the concept of alveolar bone bending by or-thodontic forces!2,13

III. HISTOMORPHOLOGY OF TOOTHMOVEMENT

A. Observations by Light MicroscopyThe pioneering work of Sandstedt and Op-

penheim opened the door for comprehensive ef-forts to explore in detail the morphologicalchanges in the stressed PDL. For over 4 decades,Reitan'4-'9 spearheaded this thrust with authorityand confidence. The strength of his work wasderived primarily from the extensive use of hu-man material, whereby teeth that were to be ex-tracted for orthodontic reasons were subjected toa variety of orthodontic force systems, i.e., light,heavy, continuous, intermittent, tipping, andtranslatory. At the end of the experimental pe-riod, the teeth were removed together with theirsurrounding tissues, and processed for histologic

evaluation. Moreover, Reitan studied paradentaltissues of animals subjected to orthodontic forces,particularly dogs and monkeys, exploring the ef-fects of age, function, type of bone, force mag-nitude, duration, and direction, on the morpho-logical characteristics of the tissues. He concludedthat PDL cells in sites of tension proliferate, andthat newly formed osteoid in these areas resorbslowly when subjected to pressure. In examiningtissues from different species,20 Reitan observedthat their responses varied, and attributed thisvariability to the differences in their structuralcomposition, i.e., alveolar bone density, fre-quency and distribution of marrow spaces, andthe cell and matrix constitution of the PDL.

The realization that the rate of orthodontictooth movement in humans varies and is unpre-dictable prompted Storey to suggest that it de-pends upon the magnitude of the applied force,21'22or the presence of hormonal fluctuations, suchas those associated with the menstrual cycle.23These clinical observations motivated Storey24-27to conduct a series of experiments in rodents inwhich he applied forces of different magnitudesto the maxillary incisors, causing lateral toothmovement and mid-premaxillary sutural widen-ing. In the rabbit and rat the teeth moved faster,as the force was increased, but as in man, thereseemed to be a range of force magnitudes thatcould be termed optimal.24 Near teeth treated withsuch a force, newly formed bone appeared moremature, while heavy forces were associated withthe formation of a highly cellular, poorly calci-fied matrix. Heavy forces caused periodontal ne-crosis and other destructive changes in the PDL,while light forces appeared to be favorite whenmoving a tooth through a thin plate of bone, asapposition of bone on the labial surface seemedto lag behind the resorptive activity on the PDLside.25 In older animals, the rate of tooth move-ment decreased, perhaps due to reduced cellularactivity.26 Based on these observations, Storey,27concluded that the process of tooth translationthrough bone consists of three different phenom-ena: bioelastic, bioplastic, and biodisruptive. ThePDL and alveolar bone, due to their fluid-fibercomposition, can be deformed elastically by ex-ternal stresses, which also evoke cellular activ-ities. When the tissue elastic limit is reached, itstarts to deform plastically, with adaptive prolif-

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erative and remodeling reactions. Prolongedforces that exceed the bioplastic limit result inbiodisruptive deformation, with ischemia, celldeath, inflammation, and repair. Clinicallyspeaking, Storey asserted that light forces withinthe bioplastic range would cause slow toothmovement, while optimal forces that cause teethto move faster are within the boundaries of thebiodisruptive range.27 He concluded that withinthe optimum range, tooth movement is rapid, butthe quality ofremodeling bone is poor, increasingthe potential for relapse, once external force ap-plication ceases.

In contrast to Reitan, who was able to studyhuman paradental tissues following the applica-tion of forces to teeth, Storey's histologic workwas conducted solely on rodents. Storey ob-served that in humans teeth move at differentrates when forces of various magnitudes are used,and he used rodent incisors to explore the reasonsfor this phenomenon. While generally Storey'sobservations correlated well with findings of otherinvestigators, it is doubtful whether conclusionsderived from experiments in rodents can be di-rectly extrapolated to man, due to marked phys-iological differences, as pointed out by Reitan.20Nevertheless, Storey's reports are significant, ashe emphasized the development of an inflam-matory process in the stressed PDL, even whenlight forces are being used.27

Reitan's and Storey's investigations dem-onstrated the complexity of the tissue reactionduring tooth movement. It was no longer per-ceived as a simple phenomenon of applied forcecausing the tooth to move within the PDL, lead-ing to tension and compression, and subsequentbone formation and resorption, but rather as adynamic set of events that involved profound al-terations in cellular functions and changes in ma-trix composition. Thus, histology facilitated themorphological description of changes in the den-toalveolar complex that followed the administra-tion of orthodontic forces to teeth. While beingunable to explain why alveolar bone and PDLcells are responsive to applied mechanicalstresses, or how these physical entities evoke bio-chemical responses by the cells, the histologicalinvestigations unveiled the sites of cellular activ-ity, and enabled other researchers to ask "why"and "how". These questions were explored by

the use of methods such as histochemistry, elec-tron microscopy, autoradiography, and immu-nohistochemistry. In addition, PDL and alveolarbone, recognized as the prime targets for ortho-dontic forces, were obtained from animals andhumans and were subjected to mechanical stressesin culture conditions, either in tissue form or asisolated cells.

B. Histochemical Changes Associatedwith Force-Induced Paradental TissueRemodeling

Activities of oxidative enzymes and phos-phatases were localized in the PDL of rats duringforce-induced tooth movement by Deguchi andMori28 and Takimoto et al.20'30 They caused toothmovement by placing a piece of stretched rubberbetween the first and second molars, forcing theteeth to move in opposite directions. (Thismethod, introduced by Waldo and Rothblatt,31was later adopted by a number of investigatorswho used rats as experimental animals in study-ing tooth movement. It does not allow measure-ments of force magnitude, and the rubber piecescan traumatize gingival and periodontal tissues.)They reported on an increase in the number ofosteoclasts displaying high succinic dehydroge-nase activity in PDL pressure zones after 24 h.In contrast, they observed no changes in the dis-tribution of acid phosphatase and lactate dehy-drogenase in the stressed PDL.

Rats were also used by Lilja et al.32 to studythe distribution and activity of a number of en-zymes associated with alveolar bone resorption.One maxillary molar in each rat was moved bu-cally by light (5 g) or heavy (36 g) forces gen-erated by a spring attached to the incisors foreither 10 h or 1, 3, 4, or 6 d. The activities ofacid phosphatase increased in PDL cells incompression zones, as well as in adjacent gin-gival cells and alveolar crest periosteum. Stainingfor acid phosphatase also increased in marrowcells and in osteocytes near the PDL pressurezone. Lactate dehydrogenase activity, a markerof vital cells, disappeared from areas of PDLpressure ("hyalinized zones"), where cells ap-parently died in both cases of light and heavyforces. Interestingly, prostaglandin synthetase

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activity was found in alveolar bone marrow cellsand in gingival cells, but not in PDL cells of therat. Unfortunately, quantitative analysis of thedata in this study was impossible, because eachgroup consisted of only one animal.

In a related experiment, Lilja et al.33 exam-ined the PDL pressure zone of human premolarsfollowing force application for 1 to 30 d. Dilatedblood vessels were seen near the hyalinized zonethroughout the experiment. Intense activities ofarylsulfatase and prostaglandin synthetase andmoderate activity of aminopeptidase M werefound in macrophages degrading the hyalinizedzone. Adjacent osteoclasts displayed intense ac-tivity for succinic dehydrogenase and acid phos-phatase. Again, each treatment time group con-sisted of one to two subjects, precluding statisticalanalysis of the data.

The generation of osteoclasts in the com-pressed PDL did not escape attention, leadinginvestigators to examine this zone in an effort toelucidate specific histochemical aspects of boneremodeling. Kurihara and Enlow34 stained ratmaxillary second molar sections with rutheniumred, which demonstrates the presence of glycos-aminoglycans (GAG). In areas of alveolar boneresorption, reattachment of the PDL seemed tooccur as a result ofGAG secretion by fibroblastsand osteoblasts, serving to link new and old col-lagenous fibers. Here, too, quantitation was im-possible due to inadequate sample size. In con-trast, Martinez and Johnson35 were better able toassess the effect of orthodontic forces on alveolarbone GAG content in rats. They moved maxillarymolars in groups of four rats, treated for 1, 3, or5 d with a spring (25 g). They found that GAGstaining in alveolar bone at PDL tension areas(achieved with alcian blue) increased at day 3.However, in an external control group that re-ceived an inactive spring, the GAG staining waslighter than in the untreated side of the maxillaof the rats treated with an active spring. Thus,this well-planned experiment demonstrated theneed for a control group in which the orthodonticappliance remains inactive.

Compression zone osteoclasts in rats werealso the targets of an investigation by Noda,36who sought to determine the effect of calcitoninon osteoclastic cytochrome c oxidase activity.First, maxillary molars were moved lingually with

a spring for periods of time ranging from 15 minto 72 h. Enzymatic activity was localized in mi-tochondria of osteoclasts by electron microscopy.Calcitonin injections caused an early reductionin the number of mitochondria and enzymaticactivity in detached osteoclasts, but this effectwas abolished 72 h after calcitonin administra-tion. These results demonstrate that locally in-duced bone resorption may be affected by a bone-seeking hormone.

The above-mentioned histochemical studiesdid not, for the most part, produce quantifiabledata. Nonetheless, they painted a picture of en-zymatically active cells, engaged in the remod-eling of the stressed PDL and alveolar bone. Inareas of PDL compression, oxidative enzymesand proteinases were localized in osteoclasts andin macrophages removing necrotic tissue fromhyalinized zones, demonstrating heightened met-abolic rates in cells involved in alveolar boneresorption and degradation of PDL matrix anddead cells. Further details on the activities ofthese cells, as well as those located in PDL ten-sion sites, are derived from experiments in whichelectron microscopy was utilized as the investi-gative tool, as discussed in the following section.

C. Ultrastructural Changes in ParadentalTissues During Tooth Movement

Despite Reitan's reported observation'9 thatparadental tissues of the rat are different thanthose of man in many respects, as, for example,morphologically and physiologically, rats re-mained the experimental animal of choice intransmission electron microscopic (TEM) studiesof paradental tissues during tooth movement.Rygh and Selvig37 described finding degradationproducts of erythrocytes in enlarged blood ves-sels and in the extravascular space of the com-pressed PDL. The tension site in the PDL wasstudied by Ten Cate et al.,38 who later also ex-amined stretched cranial sutures in rats.32 In bothPDL and suture they observed that fibroblastswere apparently engaged in synthesizing as wellas degrading collagen. Fibroblasts entering areasof matrix disruption were termed "pioneers", aterm introduced earlier by Rygh40 for identifyingcells entering hyalinized zones in the compressed

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PDL. In the latter site, Rygh was undecided onwhether these phagocytic cells were primarilymacrophages or fibroblasts. In examining PDLtension zones in rats that had been subjected toforce application (5 to 10 g) to a maxillary molar,Rygh41 observed distended blood vessels and mi-totic PDL cells. Spaces appeared in the stretchedPDL, which were filled with flocculent material.Collagen fibers were primarily oriented in thedirection of tension, but many nonoriented fi-brils, as well as elastic-like (oxytalan) fibers, werevisible. The latter were detected earlier byEdwards42 in dogs.

Teeth are attached to the alveolar bone withembedded PDL fibers (Sharpey's). Martinez andJohnson43 examined this attachment in rats usingscanning electron microscopy (SEM). They re-ported that 5 d of tooth movement significantlyreduced the diameter of the attached fibers, sug-gesting a reduction in the mechanical strength ofthe PDL. Kurihara and Enlow44 used TEM in anattempt to elucidate the nature of the attachmentofPDL fibers to bone during resorptive activities.They concluded that the most prevalent type ofattachment in resorptive sites is adhesive in na-ture. It consists of a layer of ground substance,deposited by fibroblasts on the naked surface ofrecently resorbed bone. Later, collagen fibrils aresecreted into this layer, coalescing into fibers. Inthis fashion, partially released old bone matrixfibers intermingle with the newly formed PDLfibers.

Since Kethcham's report in 1927 that ortho-dontic treatment is associated with radiographicevidence of significant dental root resorption inmany moving teeth, this phenomenon was ex-plored by numerous investigators in man and ex-perimental animals using light microscopy, TEM,and SEM. Via SEM, Kvam45 examined humanpremolars that had been exposed to orthodonticforces. After 5 d of treatment, small areas of rootresorption were found on the margins of the com-pressed PDL of all teeth, and after 25 d all treatedteeth displayed resorption lacunae penetratingthrough the cementum into the dentin. Extensiveexternal root resorption was observed in the teethof patients whose palates had been expanded rap-idly by Barber and Sims46 and by Langford andSims.47 In this procedure, heavy forces were ap-plied for about 14 d to teeth anchoring the ex-

pansion device, and the teeth were then retainedin their new position for a few months to allowbone to fill the expanded mid-palatal suture. Allanchor teeth exhibited root resorption lacunae,and the degree of resorption was directly relatedto the length of the retention period. Repair ofroot defects by cellular cementum was observed,but with little evidence ofPDL fiber reattachment.

Using light microscopy and TEM, Rygh48investigated the PDL compression zones in ratswhose molars were subjected to orthodontic forces(5, 10, or 25 g) for 2 h to 28 d. Root resorptionlacunae were seen near the hyalinized zone, inclose proximity to a rich PDL vascular network.Rygh suggested that root resorption might be aside effect of the cellular activity associated withthe removal of the necrotic tissue of the hyalin-ized zone, and described the removal of the ce-mentoid layer as the elimination of the defenseagainst resorption in an area that is strongly pro-resorptive. This proposed association betweenroot resorption and PDL injury was supportedrecently by the results of an experiment in ratsby Nakane and Kameyama.49 In that study thegingiva and PDL of a maxillary molar were in-jured repeatedly three times at 3-h intervals, byinsertion of a 2-mm-long needle. Root resorptiondeveloped within 1 d near the traumatized, in-flammatory PDL and continued through the 21d experimental period, with concomitant repairby cementoblasts.

Since mineralized tissues remodel under theinfluence of systemic and local factors, it wassuggested that factors associated with mainte-nance of calcium homeostasis might regulate theactivity of root resorbing cells. To test this hy-pothesis, tooth movement was performed by re-searchers in hypocalcemic rats. Goldie and King50created calcium deficiency in adult lactating ratsand applied 60 g force to a maxillary molar for1 to 14 d. The teeth in these rats moved signif-icantly faster than in the control animals, as theirbones underwent extensive resorption. However,SEM measurements determined that the extentof root resorption was decreased in the calcium-deficient rats, suggesting that bone resorting cellsare more responsive to bone seeking hormonesthan cells that resorb roots of teeth. In a morerecent experiment, Engstrom et al.51 moved apartmaxillary incisors in growing rats (30 d old) who

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were being fed a calcium- and vitamin-D-defi-cient diet. Using light microscopy, they deter-mined that root resorption in the moving teethwas enhanced in the hypocalcemic rats, in con-trast to the finding of Goldie and King.50 Thisdiscrepancy may be a result of the age differencebetween the two groups of hypocalcemic rats, aswell as because molars were tested in one study,while incisors were examined in the other.

D. Autoradiographic Examination of thePDL During Tooth Movement

The PDL contains a mixed population of cellsthat can synthesize or degrade bone, cementum,and the nonmineralized PDL itself. Some of themetabolic processes associated with these activ-ities have been investigated by the use of auto-radiography. In this method, radiolabeled aminoacids are injected into animals, and their time-related location is determined by exposing tissuesections to radiation-sensitive photographicemulsions. The resulting silver-bromide saltcrystals that form over radioactive sites can belocalized microscopically. In tooth movement-related studies, investigators utilized tritiatedproline (3H-proline) to study the kinetics of col-lagen synthesis and tritiated thymidine (3H-Tdr)to study cell proliferation.

Garant and his collaborators52-55 have studiedextensively the process of collagen remodelingby PDL fibroblasts. They administered 3H-pro-line into mice and rats to determine the patternof collagen synthesis by PDL fibroblasts by usingboth light microscopy and TEM. They describedPDL fibroblasts as being elongated, polarizedcells, with the nucleus positioned at one end andthe cytoplasmic and secretory part at the otherpole. Fibroblasts were found to be distributedevenly throughout the rodent PDL, and to migratebetween the fibers, interacting during motion withadjacent matrix and cells. This motion appearsto be facilitated by cellular microfilaments (actinand myosin), and by attachment to the matrixwith glycoproteins (mostly fibronectin).56 Garantand Cho concluded that in tooth movement, PDLfibroblasts in tension sites express the phenotype

of actively migrating and matrix-secreting cells.Cells in the normal PDL proliferate and die

regularly,57 but these events are markedly in-creased during tooth movement.58 Proliferativeactivities in the mechanically stressed rat PDLwere studied extensively by Roberts et al.59-66 andby Yee et al.67'68 Uptake of 3H-Tdr by PDL cellsin tension sites was increased significantly within2 h of the insertion of an elastic material betweenthe first and second maxillary molars. Most ofthe mitotic activity occurred near the bone andthe middle of PDL, but not near the dental root.Some of the newly divided cells appeared to mi-grate in the direction of the alveolar bone, per-haps because they were preosteoblasts.61 Basedon these observations, Roberts and his associatesconcluded that stretching the PDL causes G,-arrested cells to enter mitosis, while G1 cells arestimulated to start synthesizing DNA. The lattercells are readily labeled by 3H-Tdr.

In an effort to identify and classify PDL cellsat different stages of differentiation, Roberts andCox69 resorted to measurements of nuclear vol-ume in these cells. This tedious method revealedthat the nuclei of osteoblasts are larger than thoseof fibroblasts, a fact that can be used to identifycommitted osteoprogenitor cells. While fibro-blasts were found predominantly near PDL bloodvessels, osteoblastic progenitors were locatedfurther away from the vessels and closer to thebone and cementum surfaces. These reports cre-ate the impression that the PDL preosteoblasticpopulation resides solely within the boundariesof the PDL. However, McCulloch et al.70 andMcCulloch and Heersche71 have attracted atten-tion to the finding that in mice many alveolarbone marrow spaces are directly connectedthrough vascular channels with the PDL. More-over, frequent injections of 3H-Tdr into largegroups of mice, and subsequent autoradiographicexamination of their mandibles revealed that theendosteal spaces contain many labeled progenitorcells. Thickened areas of cementum were foundopposite openings of these channels in 64% ofthe examined specimens. Although it is presentlyunknown whether progenitor cells that originatein alveolar bone marrow spaces participate in thePDL response to mechanical stress, it is temptingto speculate that such an association indeed exists.

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E. Tooth Movement: Visible TissueChanges

Clinicians realized 2000 years ago (perhapsearlier) that teeth can be moved from one positionin the mouth to another by being subjected topersistent mechanical forces. Merely 250 yearsago, Hunter made the first attempt to explain thebiological basis for this dental movement. With-out having seen the involved tissues magnifiedby a microscope, he postulated that force-inducedtooth movement was facilitated by what we mayterm today as bone remodeling. This notion wasverified in the early years of the 20th century bySandstedt, who described in detail the effects ofmechanical force on the PDL-alveolar bone in-terface. His work illustrated clearly that ortho-dontic tooth movement is made possible by theresorption of alveolar bone near sites of compres-sion in the PDL, while bone apposition occurswhere the PDL is stretched. A controversy erupteda few years later when Oppenheim suggested thatthe entire alveolar bone near a moving tooth re-models, including endosteal surfaces. Most, ifnot all, of the investigators who were engaged inexploring this issue during the first half of the20th century overwhelmingly supported Sand-stedt's hypothesis because, clearly, the most dra-matic initial histologic changes could be seen inthe stressed PDL and its immediate borderingmineralized tissue surfaces, bone and dental root.Reitan, whose comprehensive histologic explo-rations spanned over 4 decades into the secondhalf of this century, reported on bone remodelingin alveolar bone marrow spaces and gingival peri-osteum, both at a distance from the stressed PDL.These observations appeared to support Oppen-heim's transformation hypothesis, and compelledReitan to accept the century-old proposition ofFarrar that orthodontic forces bend the alveolarbone. In the late 1960s Baumrind succeeded indemonstrating that such a bending effect indeedtakes place, while others have measured spikesof altered electric potentials in teeth, PDL, andalveolar bone that had been subjected to ortho-dontic forces.

The search for an optimal orthodontic force,a force that would be most efficient in movingteeth, led two investigators who examined par-adental tissues microscopically to make contrast-

ing recommendations. Schwarz warned againstusing "heavy" forces, forces that occlude PDLcapillaries and thus can damage the tissue. How-ever, Storey recommended utilizing forces thatdo cause damage to the PDL, biodisruptive forcesthat introduce inflammation into this tissue. Healso showed that within a certain range, toothmovement could be accelerated concomitantlywith an elevation in force magnitude. Like Rei-tan, Storey associated slow tooth movement inadults with a slow rate of cellular activity.

With the advent of electron microscopy, de-tailed information emerged in the last 2 decadeson the ultrastructure of dento-alveolar tissues.Cells and matrices were studied in great detailby the use of TEM and SEM. Moreover, histo-chemical and autoradiographic investigations shednew light on biochemical events that occurred inthe dento-alveolar tissue complex during normalexistence and while under altered states of me-chanical stress. It became evident that cells thatremodel the dento-alveolar complex are equippedwith an elaborate system of cytoplasmic organ-elles that enable them to synthesize and secretematrix components and the enzymes that partic-ipate in the degradation of this matrix. Garantand Ten Cate and their associates demonstratedthat PDL fibroblasts can readily remodel the ma-trix, as well as migrate through it, while Jee andRoberts and their collaborators identified preos-teoblasts both in the PDL, following their cellcycle kinetics, and through the stretched PDL.In the compressed PDL, Rygh, Kvam, and othershave identified macrophages that seemed to re-move necrotic tissue.

Taken together, the above microscopic stud-ies on both light and electronic levels have de-scribed in great detail morphological changes,and some fundamental physiological alterationsthat seem to occur in dento-alveolar tissues dur-ing tooth movement. However, with the excep-tion of the proponents of the bone bending hy-pothesis, none of the above investigations haveaddressed the question of the mechanism of trans-duction of physical stimuli to biological reac-tions. Those who advocated the idea that bentbone generates electric potentials proposed thatthese potentials somehow stimulate cells in a me-chanically stressed area by causing structural andenzymatic changes in the cellular plasma mem-

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brane. However, interest in this question has in-creased rapidly in recent years, far beyond theboundaries of orthodontic tooth movement.Moreover, it appears increasingly evident that theregulation of cellular functions in most, if notall, tissues is under the influence of local andsystemic factors that are derived from the en-docrine, nervous, and immune systems. Conse-quently, Section IV reviews evidence in supportof the hypothesis that dento-alveolar tissue re-modeling during tooth movement is an outcomeof cellular activities that are regulated by inter-actions between physical distortions and locallydistributed humoral factors that act as endocrines,paracrines, or autocrines.

IV. MECHANISMS OF CELLULARSTIMULATION IN TOOTH MOVEMENT

A. The Effect of Mechanical Stress onCells

Cells of all kinds are subjected at one timeor another to compression, stretch, and shearing.There is growing evidence that most cells haveion channels that are potentially capable of reg-ulating active and passive variations in cellularmechanics. According to Morris,72 these ionchannels are mechanosensitive, i.e., their open-state probability depends on stress at the mem-brane. Such channels were postulated decadesago as a means of mechanoelectrical transductionin muscle and nerve cells. However, it now seemsthat most other cell types have such channel com-ponents in their membranes. These channels areubiquitous, occurring at uniform density, on theorder of 1/jim2 and in every cell.73-75 Calciumions may enter cells in significant amounts throughthese channels.76-78 According to Morris and Sig-urdson,79 tensions generated in patch electrodesto activate stretch sensitive channels are of thesame order of magnitude as those measured inmigrating fibroblasts.80

Cell membrane tension may result from in-tracellular osmotic changes, contraction of cy-toskeletal elements, or physical changes in theextracellular matrix. In 1985, Ingber andFolkman81 constructed three-dimensional cellmodels comprised of a discontinuous array of

compression-resistant struts, pulled open by con-nections with tension elements. The stability ofsuch a structure depends on maintenance of ten-sional integrity. Based on these models, and ob-servations of cellular behavior in vitro, these au-thors concluded that important functions areregulated by alterations in the integrity and com-position of the extracellular matrix. Interconnec-tions between mammalian cellular nuclei and theplasma membrane, as well as with the extracel-lular matrix, are through the continuous systemof cytoskeletal filaments and cell surface trans-membrane receptors. Physical forces, either thosegenerated by the cytoskeleton or in the matrix,appear to be important regulators of cell and tis-sue growth. This interaction between force andcell function was observed to exist in skeletalmyotubes,82 lymphocytes,83 arterial smooth mus-cle cells,84 osteosarcoma cells,85 and endothelialcells.78

Ingbar and Folkman81 hypothesized that ifphysical stimuli can be translated into metabolicalterations through changes of intracellular struc-ture, then mechanochemical transduction of thesesignals is most likely mediated by the structurallinkages that join the cytoskeleton with the ex-ternal milieu. In 1985, Ingber and Jamieson86proposed that the cellular mechanism of me-chanochemical stimuli is transduced into chem-ical information through local changes in ther-modynamic parameters. In this fashion, activationenergy of a reaction is produced by pressure andvolume alterations, and various chemical reac-tions and macromolecular polymerization pro-cesses can be selectively promoted or inhibitedas a result of mechanical perturbation of the cellsurface. Indeed, Joshi et al.87 have been able todemonstrate that intracellular cytoskeletal poly-merization can be modulated by mechanical forcesapplied to the cell surface in neurites. Similarchanges may be caused by cell growth factorinteractions. For instance, Herman and Pledger88reported on alterations in the distribution of actinand vinculin in fibroblasts as a result of exposureto platelet-derived growth factor. Likewise, thearrangement and function of steroid hormone re-ceptors may be very sensitive to mechanical per-turbation because they appear to be associatedphysically with the nuclear protein matrix.89-91

Nicolini et al.92 reported that a specific in-

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crease in nuclear size is necessary for S phaseinitiation, an observation that was reported earlierby Roberts and Cox69 to occur in PDL cells.Nuclear shape seems also to play an importantrole in regulating nuclear transport. For instance,Jiang and Schindler93 studied the effect of variousgrowth factors on nuclear transport, and sug-gested that changes in nuclear shape may be per-missive for delivery of growth factor-receptorcomplexes to their site of action in the nucleus.

Experimenting with mammary epithelialcells, Emerman and Pitelka94 observed that cellrounding is usually associated with inhibition ofcell growth and with promotion of cytodiffer-entiation. Thus, cells that produce specializedproducts usually appear round, a shape that mightfacilitate exposure of specific parts of their gen-ome. In orthodontic tooth movement, such trans-formations in cellular shape are readily visible inmechanically stressed paradental cells (Figures 1to 3). In unstressed PDL sites (Figure 1), alveolarbone osteoblasts appear flat, while those in areasof PDL tension (Figure 2) seem large and round.In areas of PDL compression (Figure 3), PDLfibroblasts assume a round shape. Histologicstudies by Reitan'4-16 and Rygh41 have demon-strated that activated osteoblasts in PDL tensionsites are engaged in producing a new bone ma-trix, while PDL cells in compression sites areprimarily involved in enzymatic degradation ofthe compressed extracellular matrix.

B. The Effect of Mechanical Stress onMineralized and NonmineralizedConnective Tissues

1. Regulation of Bone Remodeling InVivo by Applied Stresses

Typically, orthodontic forces are appliedcontinuously to teeth and their surrounding tis-sues. These forces evoke cellular activity, as hasbeen demonstrated by investigators who exam-ined affected tissues mircoscopically. However,it is unclear how long a force should be appliedto stimulate target cells in particular areas ofPDLand alveolar bone. Lanyon and his associates haveaddressed this issue in a series of experimentswhereby controlled strains were applied to avian

FIGURE 1. "Flat" alveolar bone osteoblasts (arrows)in a 5-[pm horizontal section of cat maxilla, stainedimmunohistochemically for cGMP. Tissue near control,nonorthodontically treated canine. B, alveolar bone; P,periodontal ligament. (Magnification x 1400.)

bone in vivo, allowing the investigators to ex-amine, radiographically, histologically, and his-tochemically, the effects of various strains onbone remodeling95-04 Their experimental modelconsisted of surgical removal of bone from bothproximal and distal epiphyses of the ulna in tur-keys and roosters, freeing the entire diaphysisfrom regular functional strains, leaving its nerv-ous and vascular supplies intact.95 Mechanicalloads are then introduced to this bone throughstainless steel pins attached to an external loadingapparatus. The operation caused removal of load-bearing by the ulna, followed by a loss of bonemass.96 This loss was prevented by 4 cycles perday of an externally applied loading regimen(10,000 to 12,000 microstrain, 0.5 Hz), for 42d. When the number of cycles was increased to36 per day, bone formation increased signifi-cantly. Static (continuous) loads had no effect in

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FIGURE 2. Alveolar bone osteoblasts (arrows) at PDLtension site after 14 d tipping force application to catmaxillary canine. Horizontal section, 5 ixm thick, stainedimmunohistochemically for cGMP. Notice the round ap-pearance of the cells. B, alveolar bone; P, periodontalligament. (Magnification x 1400.)

this preparation on bone remodeling, while sim-ilar loads applied intermittently for a total of onlya few minutes daily increased bone mass sub-stantially.97 The magnitude of the strain in theloaded bone appeared to be directly associatedwith the nature and degree of the remodelingresponse.4 Peak longitudinal strains below 0.001were associated with bone loss, while peak strainsabove this level were associated with substantialenhancement of periosteal and endosteal boneformation.98 These effects were found also inbirds suffering from calcium deficiency."

These experiments demonstrated that bonecells in vivo are very sensitive to a small numberof strain cycles daily. A maximal osteogenic re-sponse was obtained by only 72 s of load bearing.Moreover, it seemed like the creation of a staticload environment is essentially ignored as an os-

FIGURE 3. Periodontal cells in PDL compression zoneat the border of the necrotic "hyalinized zone" after 14d of tipping force application to cat maxillary canine.Horizontal section, 5 ipm thick, stained immunohisto-chemically for cGMP. Notice the enlarged size of thecells in comparison to the thinner PDL cells seen inFigure 1. (Magnification x 1400.)

teoregulatory stimulus, suggesting that functionalinfluence on bone architecture is derived solelyfrom intermittent loading. 00 Lanyon101 then pro-posed a hypothesis to explain the mechanism bywhich bone adapts to functional load bearing. Inhis opinion, the osteocytes are the most likelycandidates to sense the distribution, rate ofchange, and magnitude of strain in the bone ma-trix. Following their recognition of a change inthe strain situation, osteocytes communicate withthe bone surface cells that remodel the bone. Itseems like the important feature of strain in thisrespect is in the occurrence of an abnormallydistributed strain rather than an unusually largestrain. Evidence for osteocytic response to ap-plied stress was found in the higher level of glu-cose-6-phosphate dehydrogenase in these cells,and a sixfold increase in the number of osteocytes

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that have incorporated 3H-uridine into theirRNA.102 Furthermore, Lanyon hypothesized thatosteocytes respond not only to the transient ef-fects of mechanical strain, but also to the per-sistent effect of mechanical strain on the matrix.As a candidate for such strain-sensitive matrixcomponents, he chose proteoglycans.103 Exam-ining bone sections in a polarizing microscope,Lanyon quantified their birefringence and ob-served matrix proteoglycan reorientation follow-ing short mechanical loading. These large, highlycharged matrix molecules could be forced bystrain to attach to cell surface receptors, or passinto the cell and attach to its cytoskeleton. Sincethe proteoglycan reorientation persists for 1 to 2d, it could provide a physical basis for a "strainmemory" in bone.'03 In a more recent study,Skerry et al.104 found that reversal reorientationofbone matrix proteoglycans also occurs in vitro,not only in avian bone, but in bones from ratsand dogs as well. They attributed this moleculardistortion to strain-generated fluid flow, usuallypreferentially oriented with the direction of col-lagen fibers.

The experiments of Lanyon and his associ-ates provide an attractive explanation of the long-lasting effects of short-lived strains on bone cells.However, the exact mechanism by which pro-teoglycan reorientation could influence the activ-ity of target cells in bone remains unknown. Inorthodontics, functional appliances subject teethto intermittent forces, leading to their gradualmovement. In this situation, bone matrix distor-tion could perhaps be evoked and act in the fash-ion proposed by Lanyon and co-workers. In con-trast, fixed-appliance orthodontics resorts tocontinuous force applications. In this mode oftreatment, tissue effects often contain widespreaddamage, intimately associated with inflammatoryand reparative responses. Moreover, orthodontictooth movement is materialized by intense re-sorptive activity of alveolar bone, while Lanyonand associates' short-term strain application didnot evoke any resorptive activity, but rather ex-tensive formative function by bone cells. None-theless, the implication of bone matrix in thetransduction of mechanical stimuli to physiolog-ical responses by bone cells serves to broadenour understanding of the mechanism of cell stim-ulation by externally applied forces. This seem-

ingly critical involvement of bone matrix in theresponse of bone cells to mechanical stress canexplain, at least in part, Oppenheim's9 earlierobservation of a transformation of the entire al-veolar process during tooth movement.

2. Bioelectric Phenomena in Bone

The dependency of bone tissue integrity andmetabolism on mechanical stress has long beenrecognized. At the present time, growing evi-dence strongly suggests that osteoporosis can becurtailed or reversed by regular physical exer-cises, which subject skeletal elements to muscle-derived forces. 05106 Astronauts and animals thathave participated in space flights or in experi-ments on simulated weightlessness demonstratedcontinuous loss of skeletal tissue due to the lackof gravitational forces.107"' These observationsimply that mechanical stresses regulate the ac-tivity of skeletal cells, confirming Wolff s6 prop-osition that the structural architecture of bonedepends on the nature of the mechanical stressesapplied to it. Applying pressure and tension tochick embryo long bone rudiments in vitro,Glucksmannll2 observed in 1942 that optimal car-tilaginous tissue structure was obtained only inthe presence of mechanical stress. Experimentsof this sort, which have persisted, demonstratedthat skeletal tissues and cells can respond to ap-plied mechanical stresses in vitro, even in theabsence of other seemingly important systems,such as the nervous and vascular systems. Thus,bone loomed brightly as a self-contained tissue,whose response to mechanical stress is inde-pendent of any other tissue system. An examplefor this rather narrow approach, which attributedmost of the control of the response of bone tomechanical stress on the bone cells themselves,can be found in the proceedings of a recent con-ference on functional adaptation in bone tissue. 13Isolated bones or bone cells were presented atthat conference as being fully capable of respond-ing biochemically to applied mechanical stresses.A proposed major link in the cascade betweenthe applied force and the biological response wasstress-generated electric potentials.114,115

The concept of the inherent ability of boneto respond to applied mechanical stress was

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boosted by Fukada and Yasuda's" report in 1957that measurable electric potentials are evokedtemporally in bent bone. They investigated dryspecimens cut from femora of man and ox, andwere able to measure direct and converse pie-zoelectric effects in those bones. They concludedthat the piezoelectric effect appears only when ashearing force is applied to the collagen fibers ofthe bone lattice, causing them to slip past eachother. More recently, Marino et al."6 investi-gated the piezoelectric characteristics of collagenfilms and concluded that these effects originateat the level of the tropocollagen molecule, or inmolecules no larger than 50 A in diameter. Fur-ther support for the concept that collagen is thesource of the piezoelectric effect came from stud-ies on tendons17'118 and on decalcified bone. 19Bassett and Becker120 reported that a net negativepotential and a net positive potential appear, re-spectively, on the compression and tension sidesof bone. These phenomena were recorded laterby Cochran et al.'21 in a segment of a bovinemandible, and by Gillooly et al.12 and by Zengoet al. 122,123 in dog mandibles. Marino and Gross'24recently compared piezoelectric surface chargesof human bone with those of the cementum anddentin of whale teeth. They found that cementumand dentin were capable of producing only about12% of the surface charge produced by corticalbone, and concluded that "piezoelectricity me-diates alveolar (bone) remodeling". This is a typ-ical statement, which narrows the effect of me-chanical stress on bone to the generation of electricpotentials by the stressed collagen.

The possibility that alveolar bone is indeedbent by the application of tipping or translatoryforces to teeth was first suggested by Farrar.5This event was later confirmed by Baumrind13 inrats and by Grimm125 in humans. It led De-Angelis'26 to propose that the alteration of theelectric environment within the stressed alveolarbone may regulate differentiation of bone pro-genitor cells. A mechanism by which these po-tentials may reach the surface of bone cells wassuggested by Pollack et al. 15 According to thisconcept, bone is surrounded by an electric doublelayer in which electric charges flow in accordancewith a stress-related fluid flow. This stress-gen-erated potential may affect the charge of cellmembranes, as well as that of macromolecules

in their vicinity. Borgens,127 examining intact anddamaged mouse bones, detected endogenous ioniccurrents that he attributed to streaming potentials,rather than to piezoelectricity, due to the long(up to 30 min) current decay period. He sug-gested that the source of current in mechanicallystressed bone is cells rather than matrix. Otter etal.,128 studying dry and wet specimens of bovinetibia, concluded that while in the dry state thecurrent is primarily piezoelectric, in wet bone thedominant mechanism is streaming potentials.

Bioelectric measurements in alveolarbone'22,'23 have demonstrated that the com-pressed (concave) side of the orthodonticallytreated bone is electronegative with respect to thetension (convex) side, suggesting that negativepotentials during bone bending can generate bonedeposition, while positive potentials are respon-sible for bone resorption. However, Borgens' ex-periments in fracture sites'27 failed to find sucha correlation. Rather, his measurements showedthat current enters the lesion, where its dispersion(i.e., its pathway and density) remains unknowndue to the complexity of the distribution of mi-neralized and nonmineralized matrices.

Whichever is the source of electric potentialsin bone, it seems that these endogenous currentsare involved in bone repair, remodeling, and per-haps growth. This conclusion led numerousinvestigators129-"33 to apply weak currents to non-union bone fractures in an effort to facilitate heal-ing. Clinical successes in orthopedics promptedorthodontists to combine orthodontic force ap-plication with administration of weak electriccurrents to jaw tissues in an effort to determinewhether a synergistic effect on the rate of toothmovement would be achieved. In the first ex-periment of this kind, Beeson et al. 134 implantedelectrodes in cat mandibles and applied a 10-1pAdirect current constantly for 5 weeks. No signif-icant differences between electrically treated andcontrol animals were found, regardless of whetherthe cathode or the anode were placed in the vi-cinity of the moving tooth. This absence of aneffect seems to have stemmed from the placementof the electrodes near the apex of the movingteeth, rather than near the alveolar crest wheremost of the force-induced bone remodeling oc-curs when teeth are tipped. Different results wereobtained by Davidovitch et al. ,135 136 who applied

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a current of 20 ILA to gingiva near orthodonticallytipped maxillary canines in young adult cats. Sig-nificant acceleration of the rate of tooth move-ment was observed after 7 and 14 d of combinedapplication of force and electricity. This poten-tiating effect was explained by the placement ofthe anode very close to the site of PDL compres-sion, where alveolar bone resorption occurs, whilethe cathode was placed in close proximity to thesite ofPDL tension, where new bone is deposited.

3. Biochemical Events in Cells Affectedby Mechanical Forces In Vitro

The ability to maintain cells and organs inculture permitted investigators to subject them tomechanical stress and to monitor their responseto either tensile or compressive forces. Theseexperiments targeted specific cellular functions,such as proliferation, synthesis, and secretion ofextracellular matrix components, or the enzymesthat degrade them. The rationale for these ex-periments is derived from the assumption that invitro conditions facilitate the exposure of sensi-tive target cells to a specific stimulus, in theabsence of any other factors that might exist invivo, which would mask or "confound" the re-sponse that is observed in vitro. As discussedbelow, the situation in the intact mammalian or-ganism differs from that of the culture system inthat bone cells in the living animal may becomesubjected to mechanical stresses simultaneouslywith signal molecules derived from neighboringendothelial cells, fibroblasts, or migratory leu-kocytes.These molecules may amplify or dimin-ish the effect of mechanical stress on the shapeand cytoskeletal structure of the bone cells. Thus,for an in-depth understanding of the nature of theresponse of cells to mechanical stress it is essen-tial to study combinations of these interactions,a design that is rarely performed for in vitroinvestigations.

Among the main investigative yardsticks thatwere utilized to explore the mechanism of ac-tivation of cells by mechanical forces were cyclicnucleotides, prostaglandins, DNA, phospha-tases, and metalloproteinases. Adenosine 3',5'-monophosphate (cyclic AMP, or cAMP) andguanosine 3',5'-monophosphate (cyclic GMP,

or cGMP) have been identified as mediators ofthe effects of external stimuli on bone cells invivo'37-'39 and in vitro.'40'141 Fluctuations in thelevels of these substances have been found tooccur in bone treated by parathyroid hormone(PTH),120 calcitonin,138 and vitamin D3,139 as wellas electric currents'40 and mechanical force.'41Prostaglandins, particularly those of the E se-ries, have been associated with bone remodelingactivities resulting from malignancies,142 gingi-val inflammation,143 rheumatic joint disease,'44and fracture.'45 Thus, fluctuations in the levelsof cAMP and cGMP in mechanically stressedcells and PGE2 in their culture media were usedfrequently as indicators of cellular respon-siveness.

In 1975, Rodan et al.141 applied compressiveforces to chick cartilaginous bone rudiments andobserved a reduction in the levels of cAMP andcGMP in these cells within 15 min. Uchida etal.146 stretched rat costochondral chondrocytesfrom 1 min to 24 h, and reported an initial in-crease in cAMP content at 3 to 5 min, with adecline to control levels shortly thereafter. Theincorporation of 35S into GAG by the stretchedcells increased significantly, but their rate ofDNAsynthesis was not altered. Interestingly, whenstretched chondrocytes were incubated in thepresence of calcitonin, their cAMP levels at 24h were much higher than those of control cellsor cells treated with PTH.

In 1980, Somjen et al.'47 stretched rat em-bryonic calvarial cells for 1 to 60 min. Theyobserved sharp increases in cAMP and PGE2 lev-els in cells and media, respectively, with peaksat 15 to 20 min and subsequent declines. BothcAMP and PGE2 levels failed to increase whenbone cells were stressed in the presence of in-domethacin, a potent inhibitor of prostaglandinsynthesis. More recently, Shen et al.'48 exposedosteoblasts cultured from rat fetal calvaria to dif-ferent concentrations of PGE2, and within 10 minobserved a transient change in shape from anepithelioid to stellate, with markedly increasednumbers of gap junctions.

Rat calvarial cells were also used by Hase-gawa et al.149 in an effort to determine the effectof stretching on DNA and matrix component syn-thesis. Continuous or intermittent stretching for2 h increased both the number of DNA synthes-

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izing cells (by 64%) and the production of os-teonectin-like molecules. These results suggestthat bone cells respond to mechanical stress byincreasing their numbers and by rearranging theircontacts to neighboring structures. Similar ef-fects on DNA synthesis were obtained with aviancalvarial osteoblasts that were stretched for up to5 d by Buckley et al. 50 In addition, the stretchedcells were uniformly aligned perpendicular to thedirection of the strain field.

Compressive forces were applied to bone cellsin a number of studies, usually by compressingthe gas phase above the culture medium. In thisfashion, Klein-Nulend et al.'51 applied intermit-tent pressure to mouse calvaria for 5 d. Thistreatment increased alkaline phosphatase activityand 45Ca uptake by the calvaria, while resorptiveactivities decreased. The net result was a 16%increase in the calvarial mineral content. Similareffects were observed by these investigators fol-lowing the application of intermittent compres-sive forces (132 g/cm2, 0.3 Hz) to fetal mousemetatarsal bone rudiments.'52 They concluded thatcompression inhibits the migration and activityof osteoclasts and their precursors. Heavier, con-tinuous compressive forces (3 atm) were appliedby Ozawa et al.'53 to osteoblast-like cells, re-sulting in suppression of osteoblastic activitiesand marked enhancement of PGE2 production.

Unquestionably, the main arena of tissue re-modeling during tooth movement is the PDL. Itis the prime target of tooth-moving mechanicalforces, and has been the subject of numerousinvestigations aimed at elucidating details of thebiological response of its cells to applied me-chanical stress. Duncan et al.154 applied mechan-ical forces to mouse molars in vivo as well as invitro. After 3 to 5 d in culture, large amounts ofPGE2 and type II collagen were synthesized bythe ligaments. A substitute model for the PDLwas introduced by Meikle et al.,155 who used aspring to apply tensile stress to rabbit calvarialsutures in vitro. They reported an increase in thetissue levels of collagenase and a reduction inthe level of tissue inhibitors of metalloproteinases(TIMP) in these sutures.

Fibroblasts isolated from chick embryos weregrown and stretched on nylon meshes by Curtisand Seehar.'56 Intermittent stretching at 0.25 to1.0 Hz caused significant increases in mitotic

frequency and in the proportion of cells in Sphase. It was concluded that stress speeds up themitotic cycle of fibroblasts, rather than switchingcells from G, to S. However, Norton et al.157reported recently that tensile forces failed tochange the cytoskeletal configuration in PDL fi-broblasts (as determined by immunofluorescenceof tubulin, vimentin, and actin), suggesting thatthese cells are not responsive to tensile forces perse, but rather to injurious effects resulting fromthese forces. Human gingival fibroblasts werestretched by Ngan et al.,158 who reported that a5% increase in cellular surface area for 5 min to2 h caused significant elevations in the levels ofcAMP and PGE2 in the cells and their media,respectively. In a related study, Ngan et al.'59reported recently on similar effects in stretchedhuman PDL fibroblasts.

Despite the major role played by PDL fibro-blasts in tooth movement, a surprisingly smallnumber of investigations on the response of thesecells to mechanical forces in vitro have been per-formed to date. However, the relative ease ofobtaining these cells from extracted healthy hu-man teeth,l60 and the availability of a number ofmechanical systems that can apply various modesof compressive and tensile stresses to cells inculture promise to facilitate new experiments inthe near future. However, since the PDL fibro-blast-like cell population is heterogeneous, con-sisting of cells with differing phenotypes, and,as the PDL also includes numerous precursors ofosteoblasts as well as epithelial and endothelialcells, strict identification and phenotyping of thesecells will be required so that the data may beinterpreted more meaningfully.

Of great curiosity and perhaps importance isthe possible role of the epithelial rests of Malas-sez in tooth movement. These clusters of epithe-lial cells, left behind during the growth of thedental root, are distributed throughout the PDLin close proximity to the root surface cementumlayer. Their functional role continues to be anenigma. Reitan'7 noted that these epithelial clus-ters are eliminated from necrotic areas of com-pressed PDL and do not regenerate. He specu-lated that these cells may play a protective rolein preventing force-induced root resorption. Bru-nette et al.'61 cultured monkey epithelial cellsderived from rests of Malassez, together with

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PDL fibroblasts. They observed no junctionalstructures between epithelial cells and fibro-blasts, but in some cases islands of epithelial cellswere sandwiched between two layers of fibro-blasts. Later, Brunette et al.'62 detected largequantities of PGE and PGF in media in whichepithelial cells derived from porcine rests of Ma-lassez had been incubated. They proposed thatthese prostaglandins might participate in the reg-ulation of alveolar bone remodeling, either byaffecting bone cells directly or indirectly by in-teracting with PDL fibroblasts and endothelialcells. Support for the concept of interaction be-tween PDL fibroblasts, epithelial, and endothe-lial cells was provided by Merrilees et al. 163 Theyfound that the GAG synthesized in vitro by PDLfibroblasts is predominantly chondroitin sulfate,whereas epithelial cells produced primarily hy-aluronic acid. However, endothelial cells or theirconditioned media, when cocultured with fibro-blasts, stimulated increased GAG synthesis, par-ticularly hyaluronic acid.

The question of whether epithelial cells fromthe rests of Malassez can produce factors thatenhance bone resorption was addressed by Bireket al.'64 They cultured epithelial cells or theirconditioned media with mouse calvaria for 4 d,causing significant increases in calcium releasefrom the bones. Indomethacin inhibited this re-sorptive effect only partially, suggesting that fac-tors other than prostaglandins are synthesized bythe epithelial cells, which may account for theosteolytic effects of the epithelial cells. The factthat epithelial cells from the rests of Malassezrespond in vitro to tensile forces was demon-strated by Brunette. 65 In his experiment the num-ber of 3H-Trd labeled cells doubled after 2 h ofstretching. Moreover, stretched cells had a highervolume of filamentous structures and more des-mosomes per unit length of cell membrane thanunstretched cells.

The above review indicates that bone cells,PDL fibroblasts, and epithelial cells from the restsof Malassez respond readily to applied mechan-ical stresses. These cellular responses includeidentifiable biochemical events that span the en-tire cellular domain. The plasma membrane, thecytoplasmic organelles, the filamentous skeleton,and the nucleus all seem to participate in thephysical-to-chemical transduction process. How-

ever, one of the main foci of attention in thisinvestigative field has been the role of prosta-glandins in this process. The following sectionbriefly reviews this issue.

4. Prostaglandins and Force-InducedBone Remodeling

Since the report by Klein and Raisz'66 in 1970that prostaglandins stimulate bone resorption intissue culture, numerous publications have im-plicated prostaglandins, particularly of the E se-ries, in the response of bone cells to chemicaland mechanical stimuli. Out of the plethora ofinvestigations emerged a hypothesis, introducedby Rodan and Martin,'67 which proposed thatosteoblasts regulate the resorptive activities ofosteoclasts. This hypothesis was based upon thefindings that osteoblasts carry receptors to all thehormones involved in the maintenance ofcalciumhomeostasis, such as PTH, calcitonin, and vi-tamin D3. Osteoblasts respond to these endocrinemolecules, as well as to locally produced agentssuch as growth factors, with elevations in cAMPcontents and prostaglandin synthesis. Thus, pros-taglandins could serve as a stimulatory link or acoupling factor between osteoblasts and osteo-clasts. Applying tensile forces to cells derivedfrom mouse embryo calvaria, Binderman et al.168stimulated the production of PGE2 and cAMP bythese cells. This effect was abolished by agentsthat bind to membrane phospholipids (gentamicinand antiphospholipid antibodies), and thus re-duce their availability for enzymatic changes.They concluded that mechanical forces exert theireffect on bone cells by the following chain ofevents: activation of phospholipase A2, releaseof arachidonic acid, increased PGE synthesis,and elevated cAMP production. Taken together,these observations assign to PGE2 a central rolein the regulation of force-induced bone cell ac-tivation. This is clearly an oversimplified conceptthat may apply to cell culture conditions, in whichmany factors found in the intact, living mam-malian organism are absent. PGE was localizedimmunohistochemically in the cat PDL by Dav-idovitch et al.169'170 The application of tippingforces to cat canines for periods of time rangingfrom 1 h to 14 d caused a significant increase in

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the staining for PGE in PDL and alveolar bonecells at sites of both tension and compression,suggesting that PGE may indeed be involved inthe response ofPDL and bone cells to mechanicalstress. However, as discussed below, other localfactors with bone cell stimulation capabilities havebeen localized recently in the mechanicallystressed PDL, suggesting that prostaglandins maybe only one agent in a battery of factors thatregulate the response of cells to force.

The great emphasis in the literature on therole of prostaglandins in the response of bonecells to applied force prompted Yamasaki and hisassociates to perform a series of experiments onrats, monkeys, and humans, where PGE was in-jected into the gingiva near orthodontically treatedteeth.171-174 In rats,171 PGE, or PGE2 administra-tion increased the number of osteoclasts in me-chanically stressed alveolar bone within 12 h. Inmonkeys,'72 the rate of tooth movement doubledduring 18 d of treatment. The drawback of thisexperiment was the sample size, which consistedof two subjects. Similar results were obtained ina study on human patients. 173174 Here, PGE, wasinjected monthly for 5 months into the gingivanear orthodontically treated canines, resulting indoubling of the rate of tooth movement. In ex-plaining why they chose PGE administration asan adjunct to orthodontic tooth movement, Ya-masaki et al. stated that the rationale for the de-cision had been the reported evidence, primarilyfrom tissue-cell culture experiments, that impli-cated PGE2 in bone resorption. However, PGE2has been shown to enhance metaphyseal bonegrowth in young rats,175 and PGE1 infusion for3 weeks enhanced alveolar bone growth in beagledogs.'76 Thus, in vivo, PGE may regulate boneresorption and formation. To investigate this dualrole of PGE in bone as a stimulator of both re-sorption and formation, Nefussi and Baron'77 cul-tured 45Ca-labeled rat fetal long bones with PGE2for 4 d. This treatment caused enhanced peri-osteal osteoblastic activity (in terms of percent-age of osteoblastic surfaces), but increased os-teoclastic resorption in medullary cavities. Thus,the effect of PGE on bone cells may differ, de-pending on their location.

Pursuing this avenue further, Lee178 movedteeth in rats by inserting an elastic band betweenthe first and second maxillary molars, according

to the method of Waldo and Rothblatt,31 for pe-riods of 6 h to 5 d. In addition, the animalsreceived either local gingival injections of PGE1twice daily, or a constant systemic administrationby a mini-osmotic pump. In both cases the num-ber of alveolar bone osteoclastic lacunae in PDLpressure sites was increased significantly, com-pared to non-PGEl-treated animals, but the effectwas more pronounced in the animals receivingPGE, systemically. This finding raises the pos-sibility of administering PGE, or PGE2 system-ically during orthodontic treatment in an effortto enhance the rate of tooth movement. However,side effects of such a treatment must not be ig-nored. These effects may include diarrhea, vom-iting, corneal congestion, and phlebitis.

Another feature of prostaglandins related tobone remodeling has come to the foreground inrecent years. Prostaglandins have long beenknown as being potent mediators of the inflam-matory process in many tissues, including car-tilage and bone. This fact led to the widespreaduse of nonsteroidal anti-inflammatory drugs incombatting rheumatoid arthritis'79'180 and jaw-bone destruction due to endodontic lesions'8' andperiodontal disease.182-'84 If tooth-moving forcesindeed cause an inflammatory reaction in the PDL,then it should be expected that not only wouldprostaglandins be found there, but other inflam-matory mediators as well. The following sectionsexamine this issue.

V. REGULATION OF TOOTH MOVEMENTBY INFLAMMATORY MEDIATORS

A. The Cells and Fluids of thePeriodontal Ligament

The PDL is a soft tissue envelope separatingthe tooth from the alveolar bone. Like all otherconnective tissues, it is comprised of cells andextracellular matrix, which consists of collagenand ground substance.'85 It contains an intricatenetwork of blood vessels and nerve endings, andis very cellular. The majority of the PDL cellsare fibroblasts, but some of these fibroblast-likecells are actually osteoprogenitor cells.65 Osteo-blasts, either active or in the form of lining cells,occupy the alveolar bone surface bordering the

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PDL, while cementoblasts cover the dental rootsurface that interfaces with the PDL. Clusters ofepithelial cells, the rests of Malassez, are spreadin the PDL in the vicinity of the root surface,while capillaries are usually more numerous inthe center of the ligament and in the zone closerto the alveolar bone. Cells migrating out of thesecapillaries, such as lymphocytes, macrophages,and mast cells, can be observed throughout thePDL. Cells may also migrate into the PDL fromneighboring marrow spaces in the alveolarbone.70,71

The PDL contains an elaborate network ofneural filaments'86 that arise from the trigeminalnerve and send neural bundles through the apicalalveolar bone in a coronal direction to the gingivaand PDL. Myelinated and unmyelinated fibersare found in the PDL, some terminating as "free"nerve endings, mostly in the inner part of thePDL, while others terminate as knob-like en-largements or as coiled nerve endings. Unmye-linated fibers usually follow PDL blood vesselsand may have a vasomotor function. In this ca-pacity, PDL nerve fibers may release, whenstressed mechanically, vasoactive peptides thatregulate movement of leukocytes out ofcapillaries.

Nerve impulses resulting from tooth move-ment can be detected in afferent fibers. Theseimpulses originate primarily in the PDL and notin the dental pulp, as shown in experiments wherethe pulp had been removed.'87 Mechanical stim-ulation activates PDL fibers that are associatedwith large-sized myelinated fibers. 188 The smallerC fibers also react to mechanical forces, but witha larger magnitude or longer duration.189 In force-induced tooth movement, the PDL nerve fibersperform two main functions: transmission of no-ciceptive impulses centrally and release of neu-ropeptides peripherally. The latter (discussed be-low) may have an important role in regulatingthe local inflammatory response, primarily byinteracting with cells of the vascular system.

Examining the PDL in mouse molars, Freezerand Sims'90 observed that 88% of the blood ves-sels consisted of venules and 12% were capil-laries. Gould et al. 91 and McCulloch andMelcher'92 found that 75 to 80% of the bloodvessels were positioned in the bony portion ofthe PDL. To test the effect of orthodontic forces

(1 to 7 d) on the PDL vascular bed, Khouw andGoldhaber'93 perfused dogs and monkeys with acolloidal suspension of carbon particles. Theyreported on dilation of vessels in both areas ofPDL tension and compression, in close proximityto sites of alveolar bone apposition and resorp-tion, respectively. Examination by TEM of se-vere PDL compression sites in rats94 revealedstasis and erythrocytic breakdown, with disin-tegration of vessel walls. However, this initialresponse was followed by a repair process, typ-ified by an invasion of the hyalinized zone by afront of cellular and vascular elements. A mark-edly increased PDL vascularity was also detectedin the inflamed PDL of beagles by Jeffcoat etal.195 using an angiographic method.

In addition to providing the PDL with a va-riety of leukocytes, the vascular system also con-tributes to its fluid composition. Bien'96 thor-oughly analyzed the dynamics of PDL fluid inrelation to tooth movement, and identified threesources of fluid in the PDL: cellular, vascular,and interstitial. The latter is localized in the groundsubstance and acts as a thixotropic gel, which isjelly-like when not in motion, and flows quiteeasily under pressure. When subjected to a steadyforce, this fluid flows within the PDL out of areasof compression and into areas of tension. Thisfluid flow, which starts as soon as the force isapplied to a tooth and is maintained over ex-tended periods of time, is apparently a crucialstep in the physicochemical behavior of the PDL.This fluid motion and rearrangement signifies theonset and progress of distortion of PDL cells andfibers. This distortion of PDL, which is seenmicroscopically as widening in areas of tensionand narrowing in sites of compression, may resultin the release of vasoactive neuropeptides, ap-pearance of stress-generated potentials, and al-terations of cellular shape. Storey27 observed va-sodilation and migration of carbon particles outof capillaries in stressed PDL within 20 min ofthe application of an orthodontic force to guineapig incisors. Thus, orthodontic forces seem toevoke an early response in the stressed PDL,which encompasses fluids, matrix fibers andground substance, and cells.

The chain of events above-reported led us topropose the following scheme to describe the in-itial effects of force on paradental tissues (Figure

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4): (1) movement of fluids within the PDL; (2)gradual distortion of the PDL; (3) generation ofstreaming potentials; (4) alteration of cellularshape and ion channel permeability; (5) neuro-peptide release from nerve endings; (6) capillaryvasodilation and migration of leukocytes into ex-travascular areas; and (7) bending of the alveolarbone and generation of piezoelectric spikes.

The outcome of such events is the introduc-tion into the stressed PDL of agents derived fromcells of the nervous and immune systems. In ad-dition, products of the endocrine system are rou-tinely delivered to the PDL through the circula-tion. Thus, on the biochemical level, mechanicalforces can result in the simultaneous exposure ofPDL cells to signals from the nervous, immune,and endocrine systems, leading to intricate andfascinating interactions and cellular responses.

B. Interactions between Cells andProducts of the Nervous, Immune, andEndocrine Systems

Recent attempts to understand the mode ofregulation of cellular activities in various tissueshave resulted in a rapidly growing volume ofevidence in support of the contention that inter-actions between cells of the nervous, immune,and endocrine systems are pivotal parts of thismechanism. In our own research we have focusedon the possibility of such interactions in thestressed PDL between neurotransmitters, cyto-kines, and hormones. The neurotransmitters wetargeted are substance P (SP), vasoactive intes-tinal peptide (VIP), calcitonin gene-related pep-tide (CGRP), and methionine enkephalin (ME).The cytokines we chose are interleukin-lao and -

ORTHODONTIC FORCE

Movement of PDL fluids

Gradual Distortion of Generation of streamingPDL matrix and cells potentials that affect PDL|1~~~~~~~\\^~ ~and alveolar bone cells

Alteration of cellularshape, cytoskeletal Neuropeptide release fromconfiguration, and ion PDL afferent nerve endingschannel permeability

Capillary vasodilation,Bending of the alveolar migration of leukocytes intobone extravascular areas

Piezoelectric effects Synthesis and release ofcytokines, growth factors, PG's

FIGURE 4. Initial effects of orthodontic forces on paradental tissues.

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113 (IL-la and IL- 13), interleukin-2 (IL-2), tu-mor necrosis factor-a (TNF-a), and gamma in-terferon (IFN--y). The reason for choosing theseparticular molecules was the existing evidenceimplicating them in bone remodeling.

Incubation of human blood monocytes withSP by Lotz et al.197 induced the release of IL-1,TNF-a, and IL-6 by these cells, demonstratingrecognition of neurotransmitters by immune cells.Similar effects were seen in B lymphocytes thatwere stimulated to differentiate by SP,198 and inneutrophils that were stimulated by SP to enhancetheir oxidative metabolism. 199 ME had a biphasiceffect on the proliferation of peripheral bloodmonocytes200 and stimulated 02 release by po-lymorphonuclear cells.201

Cells of the immune system were found tosynthesize neurotransmitter-like molecules. Sub-stance P was extracted from mouse liver granu-lomas by Weinstock et al.,202 203 and its messen-ger RNA (mRNA) was localized to the granulomaeosinophils by in situ hybridization. Roth et al.24reported that the opioid precursor proenkephalinwas secreted by activated T helper cells. Cellsof the nervous system were reported to be re-active to products of immune cells.205 For in-stance, incubation of mouse anterior pituitary cellswith IL-1 induced protein phosphorylation, butwithout cAMP elevations, which appears to bean early signal for the secretion of 13-endorphin.In another experiment, Fagarasan and Axelrod206treated pituitary cells with IL-I in the presenceof norepinephrine or isoproterenol, causing anadditive effect on 3-endorphin secretion. Su etal.207 found identical steroid receptors in guineapig brain and spleen, postulating that steroidscan, in this fashion, alter the immune functionand cause psychological changes.

C. Neuropeptides and MineralizedTissues

SP, a neuropeptide present in parts of theCNS and PNS, such as C- type sensory fibersand autonomic afferents and efferents, is releasedfrom nerve endings in response to various stim-uli. Its effects in peripheral tissues include va-sodilation and an increase in capillary permea-bility. These effects may contribute to the plasma

extravasation and increased local blood flow thataccompany inflammation. Moreover, SP canstimulate histamine release from mast cells atsites of injury and inflammation. These biolog-ical capabilities turned SP into a suspected primecontributor to the pathogenesis of rheumatoid ar-thritis. In 1984, Levine et al.208 caused adjuvantarthritis in rats, and observed that the severity ofthe disease was pronounced in joints that weredensely innervated by SP-containing afferentneurons. Injection of SP into joints increased theseverity of arthritis. Lotz et al.209 incubated syn-oviocytes from human arthritic joints with SP,causing increases in PGE2 and collagenase re-lease. Yokoyama and Fujimoto210 demonstratedthat lymphocytes from rheumatoid arthritis pa-tients could be activated by SP to present highlevels of HLA markers. Monocytes of these pa-tients, when treated with SP, released highamounts of oxygen intermediates.

A significant observation was made byO'Bryne et al.,211 who injected IL-la into rabbitknees and measured SP and PGE2 in the jointfluid at 4, 24, and 48 h. By 4 h, SP was increased;it further increased by 24 h and remained elevatedat 48 h. PGE2 levels were highest at 4 h andremained elevated at 48 h. These results highlightthe intimate association, at sites of inflammation,between cytokines, neurotransmitters, and pros-taglandins. The neurogenic component of jointinflammation was demonstrated by Lam and Fer-rell, who in one experiment212 have inhibited car-rageenan-induced knee joint inflammation in ratsby denervation, while in the other,213 abolishedit by an intraarticular injection of capsaicin, a SPreleaser and inhibitor.

Immunolocalization of SP in dental tissueswas first reported by Olgart et al.,214'215 whoobserved its presence in the feline dental pulp.In a more recent series of articles, Wakisaka etal.216-2'8 reported on the distribution and originof SP in the rat molar pulp and PDL. Theyobserved SP-containing nerves along blood ves-sels, primarily in the middle and apical regionsof the PDL. During orthodontic tooth movementin cats, intense immunohistochemical stainingfor SP in PDL tension sites was seen by Dav-idovitch et al.219 within 1 h of treatment. Fur-thermore, administration of SP to human PDLfibroblasts in vitro significantly increased the

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concentration of cAMP in the cells and PGE2in the medium within 1 min.219

Another neurotransmitter that has been im-plicated recently as a stimulator of bone resorp-tion is VIP, a 28-amino acid residual peptide,which was originally extracted from porcine duo-denum.220 In vitro studies have demonstrated thatVIP stimulates bone resorption dramatically, andthat this activity is not mediated by PGE2.221Moreover, this effect of VIP involves an 8- to13-fold increase in bone cAMP levels.221 Bindingstudies222 revealed high-affinity receptors for VIPon human osteosarcoma cells. Hohmann et al.223localized VIP in nerve fibers in the periosteumof porcine rib, tibia, and vertebra. Here, VIP wastraced to sympathetic postganglionic neurons.Herness224 localized it in mouse PDL, mainly inthe apical part around the blood vessels. Duringorthodontic tooth movement in cats,22 intensestaining for VIP was localized in the compressedPDL near sites of bone resorption and in the pulpof moving teeth.

The recent discovery of CGRP by Rosenfeldet al.226 raised the interest of investigators of neuralcontrol of bone remodeling. This neurotransmit-ter was localized in small to medium diametersensory ganglion neurons and appeared to coexistwith SP.226 In the cat, local intraarterial infusionofSP orCGRP caused a concentration-dependentincrease in nasal blood flow.227 The widespr

tooth movement

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