Int..I. De\'. lJiol. 39; 273-2HO (1995)

Reactionary dentinogenesis

ANTHONY J. SMITH", NICOLA CASSIDY', HELEN PERRY', CATHERINE BEGUE-KIRN2,JEAN-VICTOR RUCH2and HERVELESOT2

'Oral Biology, School of Dentistry, University of Birmingham, Birmingham, United Kingdomand 2/nstitut de Bi%gie Medicale, Faculte de Medecine, Strasbourg, France

ABSTRACT Reactionary dentinogenesis is the secretion of a tertiary dentine matrix by survivingodontoblast cells in response to an appropriate stimulus. Whilst this stimulus may be exogenous innature. it may also be from endogenous tissue components released from the matrix duringpathological processes. Implantation of isolated dentine extracellular matrix components in unexposedcavities of ferret teeth led to stimulation of underlying odontoblasts and a response of reactionarydentinogenesis. Affinity chromatography of the active components prior to implantation and assay forgrowth factors indicated that this material contained significant amounts of TGF-B1. a growth factorpreviously shown to influence odontoblast differentiation and secretory behavior. Reactionarydentinogenesis during dental caries probably results from solubilization of growth factors. TGF-B inparticular. from the dentine matrix which then are responsible for initiating the stimulatory effect onthe odontoblasts. Compositional differences in tertiary dentine matrices beneath carious lesions inhuman teeth have also been shown indicating modulation of odontoblast secretion during reactionaryand reparative dentinogenesis.

KEY WORDS: delllill01-:flle.'iis, t'xtrarelllliar matrix, odontoblast.\, growth farlon, dollal (arit'S

Introduction

Whilst the ability of odontoblasts to react to appropriate stimuli(e.g., disease, chemicals or trauma) and secrete a tertiary dentinematrix at specific toci has long been recogni2ed, there has offenbeen little attention paid to the exact nature of the underlyingbiological processes giving rise to such responses. Recognition ofreactionary and reparative variants of tertiary dentinogenesis asbeing distinct processes has allowed consideration of the molecu-lar mechanisms underlying such pathological processes and com-parison with the primary dentinogenesis ot physiological toothdevelopment.

This paper initially considers the chronology of dentinogenesisas a foundation to investigating these molecular mechanisms andreflection as to whether reactionary dentinogenesis represents adistinct pathological process or if it constitutes one end of thespectrum ot physiological processes. Elucidation of these proc-esses will not only allow a better understanding of the behavior ofodontoblasts, but also provide an impetus tor exploitation in thedevelopment of new biomaterials for improved clinical manage.ment of dental disease with a sound scientific basis.

Chronological considerations of dentinogenesis

The odontoblast is recognized as a post-mitotic cell, whichdifferentiates at the late bell stage of tooth development. Duringdentinogenesis. the prime function of this cell is synthesis and

secretion of the extracellular matrix (ECM) components of dentineand subsequent mineralization of the matrix. Despite this secretoryrole, the morphological appearance ot the odontoblast can varywidely throughout its life cycle (Takuwa and Nagai, 1971; Fox andHeeley, 1980; Couve, 1986; Romagnoli et al., 1990). Underphysiological conditions, this morphological appearance can berelated to the secretory activity otthe cell, but pathological changeswithin the tissue can lead to morphological changes in theodontoblast. These changes may suggest far less of a relationshipbetween morphology and secretory behavior in that odontoblastsoften lose their columnar appearance under these conditions, butmay secrete a tertiary dentine matrix (albeit often dysplastic) atquite high rates (Baume, 1980).

During tooth development. the dentine secreted up until thecompletion of root formation may be defined as primary dentineand will comprise the main bulk of the circumpulpal dentine matrixin the human tooth. This forms at a rate of approximately 4 micronsper day (Schour and Poncher, 1937; Massier and Schour, 1946;Kawasaki et al., 1980), although the rate of dentine tormation isslower in the root than the crown of the tooth. Physiologicalsecondary dentine is laid down after completion of root formationand occurs at a much slower rate throughout life (Baume, 1980).The relative positions of primary and secondary dentines are

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.Address for reprints: Oral Biology, School of Dentistry, St Chads Queenswav. Birmingham 84 6NN, England. FAX:1216258S1S.

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274 A..f. Smith et al.

E

Fig. 1. Schematic illustrationof the positions of primarydentine 11°D}and physiologi-cal secondary dentine (2~DIrelative to enamel lEI in thehuman tooth.

1'C

2' C

~indicated in Fig. 1. Deposition ot secondary dentine within the pulpchamber can often be seen to occur in an asymmetric manner I

e.g.,

in molar teeth, greater deposition is otten seen on the roof and floorof the pulp chamber.

The appearance of the matrix of primary dentine indicates aregular, tubular structure and that of secondary dentine is notdissimilar, although it has been suggested that there is slightly lesstubular structure in the latter (Mjar, 1983). The matrix of dentine,however, comprises both intertubular and peritubular (intratubular)dentine, the compositions of which show some distinct differences(Baume, 1980). Peritubular dentine contains few collagen tibrils,whilst intertubular dentine has a rich meshwork ot collagen tibrilswithin it (Jones and Boyde, 1984). Dentin phosphoproteins areabsent from predentine (Jon tell and Linde, 1983; MacDougall et al.,1985; Nakamura et al.. 1985; Gorter de Vries et al., 1986; Takagi andSasaki, 1986; Rahima et al.. 1988) and appear to be more concen-trated within peritubular dentine (Aulak et al.. 1991). Such observa-tions have led to the suggestion (Linde, 1989) that two sites ofsecretion exist in the odontoblast - one at the level ot the cell bodyto produce predentine and a second at the mineralization front giving

rise to peritubular dentine. This hypothesis helps to explain many ofthe compositional differences within the matrix of dentine in differentregions. It also indicates the complexity of the secretory process indentinogenesis. The autoradiographic and ultrastructural evidencefor this hypothesis has recently been reviewed (Linde and Goldberg,1993) and whilst it lends support to the concept of two levels ofsecretion. a number of questions remain to be addressed. Forinstance. further information is needed regarding whether secretionis mediated by vesicles at both levels and, it so, how are the vesiclesdifferentially targeted at the two levels? Little understanding exists ofthe regulation of these processes, which could occur at the transcrip-tional, translational or post-translational levels. It is also important todistinguish between secretory vesicles and endocytotic material instudying odontoblast secretion.

Secretion of matrix at the level of predentine can be seen as anincreasing overall thickness of primary or physiological secondarycircumpulpal dentine depending on the chronological stage ot thetooth. Secretion ot matrix at the mineralization front (and possiblyfrom other points along the odontoblast process) will give rise to aprogressive increase in the thickness of the peritubular dentinelayer. It has not been possible to distinguish between the peritubulardentine matrix secreted at different points in the chronologicaldevelopment ot the tooth, e.g., that secreted during primary orsecondary dentinogenesis. Dentinal tubules show a tapered struc-ture due to deposition of peritubular dentine and their gradualocclusion is a feature of the aging process giving rise to scleroticdentine (Ten Cate, 1994).

Tertiary dentine is generally recognized as representing adentine matrix laid down at specitic loci ot the pulp-dentine inter-

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face in response to environmental stimuli. Such stimuli. if ofrelatively mild degree, will stimulate an increased rate of matrixsecretion by the existing odontoblasts exposed to influence. Strongerstimuli, however, will lead to death of the odontoblasts and ifconditions in the dentine-pulp complex are favorable, a newgeneration of odontoblast-like cells secreting a reparative dentinematrix will differentiate. In an attempt to recognize the differentsequences of events in these two situations, we have proposed thetollowing definitions (Lesot et al.. 1993; Smith et al.. 1994),

Reactionary dentine: a tertiary dentine matrix secreted bysurviving post-mitotic odontoblast cells in response to an appropri-ate stimulus.

Reparative dentine: a tertiary dentine matrix secreted by a newgeneration of odontoblast-like cells in response to an appropriatestimulus, after the death ot the original post-mitotic odontoblastsresponsible for primary and physiological secondary dentine se-cretion.

These tertiary dentines are schematically depicted in Fig. 2. Thefundamental point in these proposals is that the tertiary dentinesecreted (reparative or reactionary dentine) arises from two differ-ent populations of cells, i.e., the original post-mitotic odontoblastsresponsible for primary dentinogenesis and a new generation ofodontoblast-like cells, which have differentiated from pulpal pre-cursor cells as a reparative mechanism for tissue repair.

The structure of tertiary dentines can be very variable with aspectrum of appearance ranging from a regular tubular matrixwhich is virtually indistinguishable from primary dentine to a verydystrophic, atubular matrix possibly containing cells trapped withinit. Furthermore, not only is tertiary dentine laid down at the pulp-dentine interface, thereby increasing the overall thickness otcircumpulpal dentine. it can also be observed as an increased rateof peritubular dentine deposition. It is important, however. todistinguish this increased peritubular dentine deposition withintubules from intra-tubular calcifications (Frank et al" 1964; Mjarand Furseth, 1968; Selvig, 1968). The latter are rather poorly

MILDSTIMULUS

STRONGSTIMULUS

ReactionaryDentine

ReparativeDentine

Fig. 2. Schematic illustration of reactionary dentine deposition inresponse to mild (e.g.. early caries, mild abrasion/erosion) and strongIe. g.. severe cariesl stimuli respectively. Note rhat reactionary dentineis secreted by surviving post-mitotic odontoblast ceffs (white), whilstreparative dentine is secreted by a new generation of odontoblast-like cells(black) afrer death of the original post-mitotic odonrobfasts responsible forprimary dentine secretIOn.

Reactionary de11linogellesis 275

DAY 14+

Fig. 3. Schematic illustration of implantation of an

extracellular matrix fraction isolated from dentineIMP) into unexposed cavities in ferret teeth. After 14+days. sIgnificant deposirion of reactionarydentme (Ro) bythe odontoblasts (00) Immediately underlying the cavityand in direct contact through their dentmal tubufes wasobserved, (FM, filling material !Katzino/!; TO, inert teftondisc; E, enamel; 0, dentine; p, pulp). FM TD

understood phenomena, which appear to be distinct from thenormal dentinogenic events (Baume, 1980). These intra-tubularcalcifications have been reported in the translucent zone of dentinecaries (Frank et al., 1964) and in calcium hydroxide covereddentine (Mjar and Furseth, 1968) and appear to represent aphysicochemical precipitation of hydroxyapatite orwhitlockite crys-tals rather than a vital process. Such a view is supported by in vitrodissolution experiments in dentine by weak acids (Selvig, 1968) inwhich reprecipitation of the dissolved mineral salts was seen asintratubular crystalline deposits of whitlockite and hydroxyapatite.Occlusion of dentinal tubules by peritubular dentine depositionbeneath carious lesions leading to dead tract formation is a notuncommon observation (Ten Cate, 1994). Thus, the peritubulardentine matrix may comprise secretions arising from primary,secondary or tertiary dentinogenesis. Also, the order of secretionof these accumulated matrices may vary, e.g., tertiary depositionat peritubular sites may feasibly be interposed between primaryand secondary dentine secretions, although it might be expectedthat a primary, secondary, tertiary sequence would be morecommon. Thus, any consideration of tertiary dentinogenesis withinperitubular areas is complicated by the phenomenon of physiologi-cal dentinogenesis, whether it be primary or secondary, the latterof which is a normal feature of the ageing process. It is only reallyat the pulp-dentine interface that reliable distinction can be madebetween tertiary and other dentines.

Tertiary dentinogenesis: reactionary is distinct fromreparative dentinogenesis

To define a tertiary dentine as reactionary or reparative, it isnecessary to have chronological information on the previousevents within the pulp-dentine complex (Lesot et al" 1993). In thisway, it is possible to determine whether the tertiary dentine matrixhas been secreted by the post-mitotic odontoblasts arising duringnormal tooth development or by a new population of odontoblast-like cells that have differentiated after death of fhe originalodontoblasts as a result of disease or trauma. Whilst the presenceof tertiary dentine, secreted in response to a variety of stimuli, hasbeen widely described in human teeth it is very difficult to distin-guish whether it is reactionary or reparative in nature. In theabsence of the previous history of these teeth, in terms of cellbehavior in the dentine-pulp complex, it can only be concluded thata tertiary dentine of unknown origin has been secreted. Becauseof the episodic nature of denfal caries, it is likely that the tertiary

MP

dentine beneath a carious lesion may often comprise both reac-tionary and reparative dentines superimposed upon one another.Thus, at the in vivo level, it is difficult to investigate the process ofreactionary dentinogenesis in human teeth.

Animal models offer certain advantages for the examination ofreactionary dentinogenesis in vivo where time course studies onthe processes occurring can be undertaken. Implantation of alyophilized extracellular matrix fraction isolated from dentine intounexposed cavities in ferret teeth gave rise to stimulation of theodontoblasts' synthetic and secretory activity (Smith et al., 1994b).This is schematically illustrated in Fig. 3. With implantation periodsas short as 14 days, there was significant deposition of reactionarydentine at the pulp-dentine interface by the odontoblasts immedi-ately underlying the cavity and in direct contact through theirdentinal tubules. In contrast, control cavities lacking the dentinematrix components showed no evidence of reactionary dentinedeposition. Identification of the response as one of reactionarydentinogenesis by stimulation of existing odontoblasts was con-firmed by examination of teeth at early periods ot implantation (2and 5 days), which indicated that odontoblast death had notoccurred as a result of the operative procedures. The absence ofodontoblast death in these teeth is a reflection of the carefullycontrolled method of cavity preparation since such preservationmay be harder to achieve during cavity preparation under normalclinical conditions. In a recent review of the influence of variousdental materials on odontoblast activity, it was concluded that thecavity preparation procedures had a greater influence on theodontoblast activity than the materials themselves (Cox et al"1992). This is borne out by the observations that, even using thesame cavity preparation procedures as above, the response of

reactionary dentinogenesis beneath cavities filled with variousdental materials was not nearly so strong as that seen afterimplantation of the dentine ECM components (Plant et al" 1991;Tobias et al., 1991). Thus, under carefully controlled conditions itis possible to prepare a cavity in a tooth, maintain odontoblastintegrity and subsequently stimulate these odontoblasts by implan-tation of isolated dentine ECM components to secrete a reaction-ary dentine matrix.

The absence of odontoblast death in the above study onimplantation of dentine ECM components implies that reactionarydentinogenesis can result from interaction between existingodontoblasts and appropriate molecular stimuli leading to anincrease in the synthetic and secretory activity of the odontoblast.This contrasts markedly with reparative dentinogenesis, where

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276 A.J. Smilh ('I al.

CELL SECRETION, /MOLECULAR REACTIONARY

t2 STIMULUS

~')

CYTODIFFERENTIATION

'ifCELL ADHESION

'ifCEll MIGRATION

'ifCHEMOTAXIS

'ifCELL DIVISION

REPARATIVE

DENTINOGENESIS

Fig. 4. Schematic illustration of the biological events underlyingreactionary and reparative dentinogenesis. Reactionary dentinogenesisrequires only interaction between the molecular stimulus and theodontoblast for stimulation of matri>. secretion, whilst reparativedentinogenesis requires a cascade of events terminating incytodifferentiation of a new generation of odontoblast-like cells beforesecretion of matrix can occur.

odontoblast death occurs and appropriate pulpal precursor cellsare induced to differentiate into a new population of odontoblast-like cells (Smith et a/., 1990). Itis possible that similar molecules areinvolved in the initiation of reparative and reactionary dentinogenesissince the same fraction of isolated dentine ECM components cangive rise to both ofthese processes. However, the biological eventsunderlying these processes are different (Fig. 4). In reparativedentinogenesis, a cascade of events involving cell division,chemotaxis, cell migration, cell adhesion and cytodifferentiationmust occur before secretion of the matrix can take place. Reaction.ary dentinogenesis. however, requires only interaction betweenthe molecular stimulus and the odontoblast for stimulation ofsecretion of the matrix.

Atter implantation of isolated dentine ECM components (Smithet al.. 1994), the deposition of reactionary dentine at the pulp-dentine interface increased in a non-linear manner with time ofimplantation. This may have been a result ofthe active component(s)becoming limiting due to the dose-response behavior of theodontoblasts and/or degradation of the active component(s). Theresponse of reactionary dentinogenesis decreased with increasingthickness of residual dentine beneath the cavities, probably as aresult of the increased distance of diffusion from the cavity to theodontoblasts down the dentinal tubules. Thus, the distance ofdiffusion may limit odontoblast stimulation either due to degrada-tion or dose-response, This response limiting factor of the distanceof diffusion may suggest that the interaction of the activecomponent(s) occurs on the odontoblast cell body rather than onthe odontoblast process where distance of diffusion would be lesscritical.

Whilst the same fraction at isolated dentine ECM componentscan initiate both reparative (Smith et al., 1990) and reactionary(Smith et al., 1994) dentinogenesis, it is probable that a synergisticassociation of components is required to initiate the cascade of

events in reparative dentinogenesis (see paper by Tziafas in thisissue). Reactionarydentinogenesis, however, appears to be a lesscomplex biological process and may have much simpler require-ments in terms of the initiating molecule(s). To investigate thenature of the molecules involved in the initiation of reactionarydentinogenesis, we have further purified the EDT A-soluble fractionof dentine matrix components by affinity chromatography on heparin-agarose. The bound material, eluting with 0.15 M-NaCI (similaraffinity as certain members of the transtorming growth factor Bfamily) was assessed for its ability to initiate reactionarydentinogenesis and assayed for the presence of TGF-B. Atter 14days implantation in unexposed cavities prepared in ferret canineteeth (Fig. Sa), this material (containing approximately 115 pgTGF-B, per implantation site) stimulated secretion of a tubularreactionary dentine matrix at the pulp-dentine interface immedi.ately beneath the cavities (Fig. 5b). The interface between thereactionary dentine and odontoblasts was irregular and overall, thereactionary dentine often had the appearance of tinger-like projec-tions (Fig. 5c) suggesting that closely adjacent odontoblasts hadresponded to ditterent extents to the stimulation from the implantedmaterial. The overall appearance of cellular inclusions within thereactionary dentine matrix was largely the result of the arrange-ment of the finger-like projections rather than being true cellularinclusions. Deposition of reactionary dentine in the experimentalcavities was only seen in those areas where the dentinal tubuleswere in communication with the cavity indicating the role ofdiffusion of the molecules through the dentinal tubules (Fig_ 5d).Control cavities (Fig. 5e) lacking the implanted material showed noevidence of reactionary dentinogenesis.

Use of animal models appears to be the most fruitful way toexamine reactionary dentinogenesis in vivo, but recent studiesindicate that in vitro models could be valuable in this area. Slicesof rodent incisor teeth, in which the dentine-pulp complex tissuearchitecture is maintained. have been successfully cultured forperiods up to 14 days (Sloan et a/., 1994) in a semi-solid agar basedmedium and this approach also appears feasible for culture ofhuman tooth slices. Human tooth slices have also been cultured inliquid media for similar periods at time (Magloire et a/., personalcommunication). These culture models have significant potentialfor investigating reactionary dentinogenesis, where odontoblastbehavior can be examined independently of the normal inflamma-tory processes which can occur in vivo.

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Variations in the odontoblast phenotype

The morphological appearance of the odontoblast and thetubular matrix it secretes have been some of the most character-istic features of this cell. Whilst certain differences in the composi-tion of dentine and the closely related hard-tissue, bone, can beobserved, it is not clear whether these are characteristic enough onan individual molecular basis to be used as phenotypic markers.Even morphological appearance is fraught with problems since theodontoblast cell changes in appearance through its life-cycle(Takuwa and Nagai, 1971; Fox and Heeley, 1980; Couve, 1986;Romagnoli et al.. 1990) and the regularity of the tubular structurevaries trom primary to tertiary dentines. In situ hybridization hasallowed comparison of the expression of various transcripts innormal odontoblasts and odontoblast-like cells induced by variousgrowth factors (Begue-Kim et al., 1994). These studies haveshown that odontoblasts and odontoblast-like cells, which are

Reactionary dentinogenesis 277

A EDENTINE

I

I,

EDTA

IO.15M eluted c:>heparin-agarosefraction

FM TO MP

o --- >f

IFig. 5. Implantation of dentine matrix fraction (MP), purified by heparin-agarose affinity chromatography, in unexposed cavities in ferret canine

teeth. (AI Schematic illustration of implantation (FM, filling material [Kalzinol]; TO, inert teflon disc; E, enamel; 0, dentine; 00, odontoblasts; P, pulp).(B-D) Pulp-dentine interface beneath experimental cavities 14 days after Implantation of the dentine matrix fraction. !B) Significant deposition of tubular

reactionary dentine matrix (arrow) immediately beneath the cavity, x200. IC) Irregular interface between the reactionary dentine and odontoblasts withthe appearance of finger-like projections (arrows), x320. (D) Secretion of reactionary dentrne was limited to only those areas where the dentinal rubuleswere in communrcation with the cavity floor (arrows indicate outer limits of communication at cavity and pulp-dentine interface). x200. (E) Pulp-dentineinterface beneath a control cavity lacking the implanted matnx fraction and showing no evidence of reactionary dentinogenesis, x200. (S-EI Black barsat bottom left of Figs. represents 10 microns).

characteristic of primary and tertiary dentinogenesis, have thepotential for elaboration of similar matrix components. Normalodontoblasts and odontoblast-like cells induced by immobilizedTGF-B1 or BMP-2 expressed transcripts encoding tor TGF-B1 and

3, BMP-2 and -4, bone sialoprotein and osteonectin, whereaseither ubiquitous expression or no expression could be detected forTGF-B2, IGF-1 or tibronectin mRNAs. Odontoblast-like cells ob-tained in the presence of immobilized IGF-1, however, did not

278 A.J. Sm;l1I el al.

express TGF-B1 transcripts and only weakly expressed TGF-B3transcripts. Thus, use of a panel of markers may be necessary tocharacterize the phenotype at odontoblasts and odontoblast-likecells.

The changes in activity of the odontoblast throughout its life andthe stimulation of its synthetic and secretory activity in response toappropriate stimuli resulting in reactionary dentine depositionindicate that the odontoblast is a very versatile cell. Morphologicaldifferences in the tubular structure of the matrix secreted by thesecells in primary, secondary and tertiary dentines could indicatemolecular changes in these matrices. An increase in the secretionof extracellular matrix has been demonstrated in tertiary dentinebeneath carious lesions in human teeth (Lesot et al., 1993; Perryand Smith, unpublished observations) and immunohistochemicalapproaches have shown re-expression of collagen type III,fibronectin and the membrane 165 kDa fibronectin binding proteinin this tissue (Magloire et al., 1988, 1992), although these threeproteins have a transitory expression during the differentiation ofprimary odontoblasts (Lesot et al., 1990; Lukinmaa et al., 1991).Differences in the expression of phosphoproteins in primary,secondary and tertiary dentines have also been reported (Takagiand Sasaki, 1986; Aulak et al., 1991). It seems probable that thesevarious differences in matrix composition reflect modulation inodontoblast activity.

To investigate this further, the EDTA-soluble collagen,noncoliagenous protein and glycosaminoglycan levels have beenanalyzed in reactionary/reparative dentines beneath carious le-sions in human teeth (Perry and Smith, unpublished observations).The tertiary dentine specimens contained up to four times greaterlevels of these components compared with primary dentine. How-ever, these levels were not uniformly raised for the three classes ofmatrix components suggesting that there had been differentialstimulation at the synthetic/secretory pathways for these compo-nents. Thus, it is clear that modulation of odontoblast activity canoccur and that variation in expression of the different ECM compo-nents can result. Attempts to relate these compositional changesto the degree of regularity of the tissue tubular structure indicatedlittle relationship between these parameters. It is now important todeterminethe molecular basis otthis modulation of the odontoblastsand to investigate whether molecular stimuli can act on differentaspects of the cell's synthetic/secretory processes.

Molecular basis of odontoblast stimulation in reaction-ary dentinogenesis

Experiments on the implantation of isolated dentine ECM com-ponents reported above (Smith et al., 1994) have demonstratedthat interaction between these components and odontoblastsresults in stimulation of the synthetic/secretory activity of thesecells. Contact between odontoblasts and their extracellular matrixappears to be important in the maintenance of their phenotype aswell. The phenotypic morphology of these cells when cultured canonly be preserved as long as contact is maintained between thecells and dentine matrix (Munksgaard et al., 1978; Heywood andAppleton, 1984). If the odontoblasts are removed from the toothand cultured in isolation, they take on the appearance of a fibroblasticmorphology. It is possible to replace the mature dentine matrix bypreparations of isolated dentine ECM components as demon-strated by maintenance of odontoblast differentiation when dentalpapilla were cultured in contact with such preparations for 4 days(Lesot et al., 1986).

The nature of the molecules in the isolated dentine ECMfractions which can interact with odontoblasts and stimulatetheir activity may be varied, although involvement of growthfactors seems probable. The heparin-agarose bound material,which stimulated reactionary dentinogenesis as described inthe implantation experiments above contained significantamounts of TGF-B1, but not TGF-B2 or 3. Culture of dentalpapillae with TGF-B1, or the closely related BMP-2, stimulatedextracellular matrix secretion by these cells (Begue-Kim et al.,1992). This pattern at cell behavior was ditferent to that ob-served when these growth factors were added with potentiatingmolecules, such as heparin, when they were shown to becapable of inducing the ditferentiation of odontoblast-like cells(Begue-Kim et al., 1992).

There are many reports of the effects of TGF-B, and relatedmembers at this growth factor tamily, on matrix synthesis. BMP-2has been reported to influence the activity of cultured osteoblasticMC3T3-E1 cells, where it stimulates alkaline phosphatase andcollagen synthesis (Takuwa et al., 1991). TGF-B modulates thesynthesis of collagens (Ignotz and Massague, 1986; Yu et al.,1991) and that at proteoglycans (Rapraeger, 1989; Yu et al.. 1991).Clearly, TGF -B and related molecules can have a marked intluenceon extracellular matrix synthesis, but it is important to note that,depending on its concentration, TGF-B can stimulate matrix syn-thesis orresorption (Tashjian et al., 1985; Hock et al., 1990). Thus,the control mechanisms on the activity of these growth factors is ofparamount importance in determining their effects on cell behaviorin the tissue.

There are a number of levels at which control of growth factoractivity could occur within the tissue. At the level of interaction withthe cell, whilst considerable information exists on TGF-B receptors(Boyde et al., 1990) in general, little Information exists on thesereceptors in relation to odontoblasts. The presence ot theextracellular matrix proteoglycan, decorin, has been recently re-ported in dentine matrix (Lesot et al., 1994). Decorin not only bindsTGF-B, but can also neutralize the activity of this growth tactor. Thetime course for initiation of reactionary dentinogenesis whenisolated dentine ECM components are implanted in unexposedcavities (Smith et al., 1994) suggests that the interactions of theactive molecule(s) occurs on the cell body rather than the processof the odontoblast. Further studies are now needed to elucidate thenature of the odontoblast growth factor interactions.

Control of growth factor activity can also occur by virtue of theavailability of the growth factor. Finkelman et al. (1990) havedemonstrated the presence of a number of growth factors indentine, including TGF-B, and have suggested that the responseof odontoblasts to dental caries may be mediated by release ofthese growth factors from dentine during demineralization bybacterial acids. Our own studies have shown the presence of TGF-B1, 2 and 3 in dentine and that these molecules may be bound upin different extracellular matrix tissue compartments (Smith et al.,unpublished observations). A proportion of these molecules can bereleased by demineralization, but some appear to be tightly asso-ciated with the collagenous matrix and require collagenase diges-tion to release them. It is not clear yet as to whether thesemolecules are present in an active or latent state, but furtherinvestigations are now required to understand the role of thesemolecules and whether they participate in physiological tissuefunction (perhaps helping to maintain the odontoblast phenotype)or if they are only available/exposed in pathological conditionswhen they can initiate a response of tertiary dentinogenesis.

Conclusions

Deposition of reactionary dentine by post-mitotic odontoblasts,which have ditterentiated during tooth development. can arise atthe pulp-dentine interface and at a peritubular/intratubular locationin response to an appropriate stimulus. Some of this reactionarydentine may be a teature of normal physiological processes, suchas aging, although it is not clear as to what extent pathologicalprocesses such as dental disease, tooth wear etc. contribute to this"physiological" appearance. Probably the majority of reactionarydentine arises in response to dental disease. Traditional views ofthe acid generated from plaque bacterial metabolism being thestimulus to the odontoblasts for reactionary dentinogenesis havelittle evidence to supporfthem. It is likely that plaque bacterial acidssoiubilize growth factors, TGF-B in particular, as they permeatedentine and that it is the solubilized growth factors that areresponsible for initiating the stimulatory ettect on the odontoblaststo secrete reactionary dentine.

AcknowfedgmentsThe research was supported by the Commission of the European

Communities SCIENCE Programme Contract no. SC1 ~-CT91-0680(WNSN).

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