Reactionary Dentinogenesis

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

I Reactionary dentinogenesis

I ANTHONY J. SMITH", NICOLA CASSIDY', HELEN PERRY', CATHERINE BEGUE-KIRN2, JEAN-VICTOR RUCH2and HERVELESOT2 I 'Oral Biology, School of Dentistry, University of Birmingham, Birmingham, United Kingdom and 2/nstitut de Bi%gie Medicale, Faculte de Medecine, Strasbourg, France

I ABSTRACT Reactionary dentinogenesis is the secretion of a tertiary dentine matrix by surviving odontoblast cells in response to an appropriate stimulus. Whilst this stimulus may be exogenous in nature. it may also be from endogenous tissue components released from the matrix during pathological processes. Implantation of isolated dentine extracellular matrix components in unexposed I cavities of ferret teeth led to stimulation of underlying odontoblasts and a response of reactionary dentinogenesis. Affinity chromatography of the active components prior to implantation and assay for growth factors indicated that this material contained significant amounts of TGF-B1. a growth factor I previously shown to influence odontoblast differentiation and secretory behavior. Reactionary dentinogenesis during dental caries probably results from solubilization of growth factors. TGF-B in particular. from the dentine matrix which then are responsible for initiating the stimulatory effect on I the odontoblasts. Compositional differences in tertiary dentine matrices beneath carious lesions in human teeth have also been shown indicating modulation of odontoblast secretion during reactionary and reparative dentinogenesis. I

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

secretion of the extracellular matrix (ECM) components of dentine Introduction I and subsequent mineralization of the matrix. Despite this secretory Whilst the ability of odontoblasts to react to appropriate stimuli role, the morphological appearance ot the odontoblast can vary (e.g., disease, chemicals or trauma) and secrete a tertiary dentine widely throughout its life cycle (Takuwa and Nagai, 1971; Fox and I matrix at specific toci has long been recogni2ed, there has offen Heeley, 1980; Couve, 1986; Romagnoli et al., 1990). Under been little attention paid to the exact nature of the underlying physiological conditions, this morphological appearance can be biological processes giving rise to such responses. Recognition of related to the secretory activity otthe cell, but pathological changes reactionary and reparative variants of tertiary dentinogenesis as within the tissue can lead to morphological changes in the I being distinct processes has allowed consideration of the molecu- odontoblast. These changes may suggest far less of a relationship lar mechanisms underlying such pathological processes and com- between morphology and secretory behavior in that odontoblasts parison with the primary dentinogenesis ot physiological tooth often lose their columnar appearance under these conditions, but I development. may secrete a tertiary dentine matrix (albeit often dysplastic) at This paper initially considers the chronology of dentinogenesis quite high rates (Baume, 1980). as a foundation to investigating these molecular mechanisms and During tooth development. the dentine secreted up until the I reflection as to whether reactionary dentinogenesis represents a completion of root formation may be defined as primary dentine distinct pathological process or if it constitutes one end of the and will comprise the main bulk of the circumpulpal dentine matrix spectrum ot physiological processes. Elucidation of these proc- in the human tooth. This forms at a rate of approximately 4 microns I esses will not only allow a better understanding of the behavior of per day (Schour and Poncher, 1937; Massier and Schour, 1946; odontoblasts, but also provide an impetus tor exploitation in the Kawasaki et al., 1980), although the rate of dentine tormation is development of new biomaterials for improved clinical manage. slower in the root than the crown of the tooth. Physiological I ment of dental disease with a sound scientific basis. secondary dentine is laid down after completion of root formation and occurs at a much slower rate throughout life (Baume, 1980). The relative positions of primary and secondary dentines are Chronological considerations of dentinogenesis I

The odontoblast is recognized as a post-mitotic cell, which .-th"rn.'illliOlu IIIf'd j/l tll;} Imp,.,: TCF.H. tramfollnint-:' j.!;rO\\lh fanor (1;R\II', I(;r, iw.ulin J.::l"lIh.thfaClnr:rOTA. t"thylt-IW- differentiates at the late bell stage of tooth development. During OOI1t" mllrpho~I'IH'lil' pwtl'in: I dentinogenesis. the prime function of this cell is synthesis and diallljJl('It'traan'tic acid; EC\I, t'xtran:llillar IJI;Llrix.

.Address for reprints: Oral Biology, School of Dentistry, St Chads Queenswav. Birmingham 84 6NN, England. FAX:1216258S1S. I 0214-6282/95/$03.00 e liar: f>rcu rrinlcdin Splin I

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274 A..f. Smith et al.

face in response to environmental stimuli. Such stimuli. if of relatively mild degree, will stimulate an increased rate of matrix secretion by the existing odontoblasts exposed to influence. Stronger E stimuli, however, will lead to death of the odontoblasts and if conditions in the dentine-pulp complex are favorable, a new 1'C Fig. 1. Schematic illustration generation of odontoblast-like cells secreting a reparative dentine of the positions of primary matrix will differentiate. In an attempt to recognize the different sequences of events in these two situations, we have proposed the 2' C dentine 11°D}and physiological secondary dentine (2~DI tollowing definitions (Lesot et al.. 1993; Smith et al.. 1994), relative to enamel lEI in the Reactionary dentine: a tertiary dentine matrix secreted by ~ human tooth. surviving post-mitotic odontoblast cells in response to an appropriate stimulus. indicated in Fig. 1. Deposition ot secondary dentine within the pulp Reparative dentine: a tertiary dentine matrix secreted by a new e.g., chamber can often be seen to occur in an asymmetric manner I generation of odontoblast-like cells in response to an appropriate in molar teeth, greater deposition is otten seen on the roof and floor stimulus, after the death ot the original post-mitotic odontoblasts of the pulp chamber. responsible for primary and physiological secondary dentine se- The appearance of the matrix of primary dentine indicates a cretion. regular, tubular structure and that of secondary dentine is not These tertiary dentines are schematically depicted in Fig. 2. The dissimilar, although it has been suggested that there is slightly less fundamental point in these proposals is that the tertiary dentine tubular structure in the latter (Mjar, 1983). The matrix of dentine, secreted (reparative or reactionary dentine) arises from two differ- however, comprises both intertubular and peritubular (intratubular) ent populations of cells, i.e., the original post-mitotic odontoblasts dentine, the compositions of which show some distinct differences responsible for primary dentinogenesis and a new generation of (Baume, 1980). Peritubular dentine contains few collagen tibrils, odontoblast-like cells, which have differentiated from pulpal pre- whilst intertubular dentine has a rich meshwork ot collagen tibrils cursor cells as a reparative mechanism for tissue repair. within it (Jones and Boyde, 1984). Dentin phosphoproteins are The structure of tertiary dentines can be very variable with a absent from predentine (Jon tell and Linde, 1983; MacDougall et al., spectrum of appearance ranging from a regular tubular matrix 1985; Nakamura et al.. 1985; Gorter de Vries et al., 1986; Takagi and which is virtually indistinguishable from primary dentine to a very Sasaki, 1986; Rahima et al.. 1988) and appear to be more concen- dystrophic, atubular matrix possibly containing cells trapped within trated within peritubular dentine (Aulak et al.. 1991). Such observa- it. Furthermore, not only is tertiary dentine laid down at the pulp- tions have led to the suggestion (Linde, 1989) that two sites of dentine interface, thereby increasing the overall thickness ot secretion exist in the odontoblast - one at the level ot the cell body circumpulpal dentine. it can also be observed as an increased rate to produce predentine and a second at the mineralization front giving of peritubular dentine deposition. It is important, however. to rise to peritubular dentine. This hypothesis helps to explain many of distinguish this increased peritubular dentine deposition within the compositional differences within the matrix of dentine in different tubules from intra-tubular calcifications (Frank et al" 1964; Mjar regions. It also indicates the complexity of the secretory process in and Furseth, 1968; Selvig, 1968). The latter are rather poorly dentinogenesis. The autoradiographic and ultrastructural evidence for this hypothesis has recently been reviewed (Linde and Goldberg, MILD STRONG 1993) and whilst it lends support to the concept of two levels of STIMULUS STIMULUS secretion. a number of questions remain to be addressed. For instance. further information is needed regarding whether secretion is mediated by vesicles at both levels and, it so, how are the vesicles differentially targeted at the two levels? Little understanding exists of the regulation of these processes, which could occur at the transcrip- tional, translational or post-translational levels. It is also important to distinguish between secretory vesicles and endocytotic material in studying odontoblast secretion. Secretion of matrix at the level of predentine can be seen as an increasing overall thickness of primary or physiological secondary circumpulpal dentine depending on the chronological stage ot the tooth. Secretion ot matrix at the mineralization front (and possibly from other points along the odontoblast process) will give rise to a progressive increase in the thickness of the peritubular dentine Reactionary Reparative layer. It has not been possible to distinguish between the peritubular Dentine Dentine dentine matrix secreted at different points in the chronological development ot the tooth, e.g., that secreted during primary or Fig. 2. Schematic illustration of reactionary dentine deposition in secondary dentinogenesis. Dentinal tubules show a tapered struc- response to mild (e.g.. early caries, mild abrasion/erosion) and strong ture due to deposition of peritubular dentine and their gradual Ie. g.. severe cariesl stimuli respectively. Note rhat reactionary dentine occlusion is a feature of the aging process giving rise to sclerotic is secreted by surviving post-mitotic odontoblast ceffs (white), whilst dentine (Ten Cate, 1994). reparative dentine is secreted by a new generation of odontoblast-like cells Tertiary dentine is generally recognized as representing a (black) afrer death of the original post-mitotic odonrobfasts responsible for dentine matrix laid down at specitic loci ot the pulp-dentine inter- primary dentine secretIOn. Reactionary de11linogellesis 275

DAY 14+

Fig. 3. Schematic illustration of implantation of an extracellular matrix fraction isolated from dentine IMP) into unexposed cavities in ferret teeth. After 14+ days. sIgnificant deposirion of reactionarydentme (Ro) by the odontoblasts (00) Immediately underlying the cavity and in direct contact through their dentmal tubufes was observed, (FM, filling material !Katzino/!; TO, inert tefton FM TD MP disc; E, enamel; 0, dentine; p, pulp). understood phenomena, which appear to be distinct from the dentine beneath a carious lesion may often comprise both reac- normal dentinogenic events (Baume, 1980). These intra-tubular tionary and reparative dentines superimposed upon one another. calcifications have been reported in the translucent zone of dentine Thus, at the in vivo level, it is difficult to investigate the process of caries (Frank et al., 1964) and in calcium hydroxide covered reactionary dentinogenesis in human teeth. dentine (Mjar and Furseth, 1968) and appear to represent a Animal models offer certain advantages for the examination of physicochemical precipitation of hydroxyapatite orwhitlockite crys- reactionary dentinogenesis in vivo where time course studies on tals rather than a vital process. Such a view is supported by in vitro the processes occurring can be undertaken. Implantation of a dissolution experiments in dentine by weak acids (Selvig, 1968) in lyophilized extracellular matrix fraction isolated from dentine into which reprecipitation of the dissolved mineral salts was seen as unexposed cavities in ferret teeth gave rise to stimulation of the intratubular crystalline deposits of whitlockite and hydroxyapatite. odontoblasts' synthetic and secretory activity (Smith et al., 1994b). Occlusion of dentinal tubules by peritubular dentine deposition This is schematically illustrated in Fig. 3. With implantation periods beneath carious lesions leading to dead tract formation is a not as short as 14 days, there was significant deposition of reactionary uncommon observation (Ten Cate, 1994). Thus, the peritubular dentine at the pulp-dentine interface by the odontoblasts immedi- dentine matrix may comprise secretions arising from primary, ately underlying the cavity and in direct contact through their secondary or tertiary dentinogenesis. Also, the order of secretion dentinal tubules. In contrast, control cavities lacking the dentine of these accumulated matrices may vary, e.g., tertiary deposition matrix components showed no evidence of reactionary dentine at peritubular sites may feasibly be interposed between primary deposition. Identification of the response as one of reactionary and secondary dentine secretions, although it might be expected dentinogenesis by stimulation of existing odontoblasts was con- that a primary, secondary, tertiary sequence would be more firmed by examination of teeth at early periods ot implantation (2 common. Thus, any consideration of tertiary dentinogenesis within and 5 days), which indicated that odontoblast death had not peritubular areas is complicated by the phenomenon of physiologi- occurred as a result of the operative procedures. The absence of cal dentinogenesis, whether it be primary or secondary, the latter odontoblast death in these teeth is a reflection of the carefully of which is a normal feature of the ageing process. It is only really controlled method of cavity preparation since such preservation at the pulp-dentine interface that reliable distinction can be made may be harder to achieve during cavity preparation under normal between tertiary and other dentines. clinical conditions. In a recent review of the influence of various dental materials on odontoblast activity, it was concluded that the Tertiary dentinogenesis: reactionary is distinct from cavity preparation procedures had a greater influence on the reparative dentinogenesis odontoblast activity than the materials themselves (Cox et al" 1992). This is borne out by the observations that, even using the To define a tertiary dentine as reactionary or reparative, it is same cavity preparation procedures as above, the response of necessary to have chronological information on the previous reactionary dentinogenesis beneath cavities filled with various events within the pulp-dentine complex (Lesot et al" 1993). In this dental materials was not nearly so strong as that seen after way, it is possible to determine whether the tertiary dentine matrix implantation of the dentine ECM components (Plant et al" 1991; has been secreted by the post-mitotic odontoblasts arising during Tobias et al., 1991). Thus, under carefully controlled conditions it normal tooth development or by a new population of odontoblast- is possible to prepare a cavity in a tooth, maintain odontoblast like cells that have differentiated after death of fhe original integrity and subsequently stimulate these odontoblasts by implan- odontoblasts as a result of disease or trauma. Whilst the presence tation of isolated dentine ECM components to secrete a reaction- of tertiary dentine, secreted in response to a variety of stimuli, has ary dentine matrix. been widely described in human teeth it is very difficult to distin- The absence of odontoblast death in the above study on guish whether it is reactionary or reparative in nature. In the implantation of dentine ECM components implies that reactionary absence of the previous history of these teeth, in terms of cell dentinogenesis can result from interaction between existing behavior in the dentine-pulp complex, it can only be concluded that odontoblasts and appropriate molecular stimuli leading to an a tertiary dentine of unknown origin has been secreted. Because increase in the synthetic and secretory activity of the odontoblast. of the episodic nature of denfal caries, it is likely that the tertiary This contrasts markedly with reparative dentinogenesis, where

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276 A.J. Smilh ('I al.

CELL SECRETION events in reparative dentinogenesis (see paper by Tziafas in this / issue). Reactionarydentinogenesis, however, appears to be a less , complex biological process and may have much simpler require- I MOLECULAR REACTIONARY ments in terms of the initiating molecule(s). To investigate the nature of the molecules involved in the initiation of reactionary t2 STIMULUS dentinogenesis, we have further purified the EDTA-soluble fraction of dentine matrix components by affinity chromatography on heparin- I agarose. The bound material, eluting with 0.15 M-NaCI (similar affinity as certain members of the transtorming growth factor B ~ family) was assessed for its ability to initiate reactionary ') dentinogenesis and assayed for the presence of TGF-B. Atter 14 I CYTODIFFERENTIATION days implantation in unexposed cavities prepared in ferret canine 'if teeth (Fig. Sa), this material (containing approximately 115 pg CELL ADHESION REPARATIVE TGF-B, per implantation site) stimulated secretion of a tubular 'if I CEll MIGRATION DENTINOGENESIS reactionary dentine matrix at the pulp-dentine interface immedi. 'if ately beneath the cavities (Fig. 5b). The interface between the CHEMOTAXIS reactionary dentine and odontoblasts was irregular and overall, the 'if reactionary dentine often had the appearance of tinger-like projec- CELL DIVISION I tions (Fig. 5c) suggesting that closely adjacent odontoblasts had Fig. 4. Schematic illustration of the biological events underlying responded to ditterent extents to the stimulation from the implanted reactionary and reparative dentinogenesis. Reactionary dentinogenesis material. The overall appearance of cellular inclusions within the

requires only interaction between the molecular stimulus and the reactionary dentine matrix was largely the result of the arrange- I odontoblast for stimulation of matri>. secretion, whilst reparative ment of the finger-like projections rather than being true cellular dentinogenesis requires a cascade of events terminating in inclusions. Deposition of reactionary dentine in the experimental cytodifferentiation of a new generation of odontoblast-like cells before cavities was only seen in those areas where the dentinal tubules secretion of matrix can occur. were in communication with the cavity indicating the role of I diffusion of the molecules through the dentinal tubules (Fig_ 5d). odontoblast death occurs and appropriate pulpal precursor cells Control cavities (Fig. 5e) lacking the implanted material showed no are induced to differentiate into a new population of odontoblast- evidence of reactionary dentinogenesis. like cells (Smith et a/., 1990). Itis possible that similar molecules are Use of animal models appears to be the most fruitful way to I involved in the initiation of reparative and reactionary dentinogenesis examine reactionary dentinogenesis in vivo, but recent studies since the same fraction of isolated dentine ECM components can indicate that in vitro models could be valuable in this area. Slices give rise to both ofthese processes. However, the biological events of rodent incisor teeth, in which the dentine-pulp complex tissue underlying these processes are different (Fig. 4). In reparative architecture is maintained. have been successfully cultured for I dentinogenesis, a cascade of events involving cell division, periods up to 14 days (Sloan et a/., 1994) in a semi-solid agar based chemotaxis, cell migration, cell adhesion and cytodifferentiation medium and this approach also appears feasible for culture of must occur before secretion of the matrix can take place. Reaction. human tooth slices. Human tooth slices have also been cultured in I ary dentinogenesis. however, requires only interaction between liquid media for similar periods at time (Magloire et a/., personal the molecular stimulus and the odontoblast for stimulation of communication). These culture models have significant potential secretion of the matrix. for investigating reactionary dentinogenesis, where odontoblast behavior can be examined independently of the normal inflamma- Atter implantation of isolated dentine ECM components (Smith I et al.. 1994), the deposition of reactionary dentine at the pulp- tory processes which can occur in vivo. dentine interface increased in a non-linear manner with time of implantation. This may have been a resultofthe active component(s) Variations in the odontoblast phenotype becoming limiting due to the dose-response behavior of the I odontoblasts and/or degradation of the active component(s). The The morphological appearance of the odontoblast and the response of reactionary dentinogenesis decreased with increasing tubular matrix it secretes have been some of the most character- thickness of residual dentine beneath the cavities, probably as a istic features of this cell. Whilst certain differences in the composi- result of the increased distance of diffusion from the cavity to the tion of dentine and the closely related hard-tissue, bone, can be I odontoblasts down the dentinal tubules. Thus, the distance of observed, it is not clear whether these are characteristic enough on diffusion may limit odontoblast stimulation either due to degrada- an individual molecular basis to be used as phenotypic markers. tion or dose-response, This response limiting factor of the distance Even morphological appearance is fraught with problems since the of diffusion may suggest that the interaction of the active odontoblast cell changes in appearance through its life-cycle I component(s) occurs on the odontoblast cell body rather than on (Takuwa and Nagai, 1971; Fox and Heeley, 1980; Couve, 1986; the odontoblast process where distance of diffusion would be less Romagnoli et al.. 1990) and the regularity of the tubular structure critical. varies trom primary to tertiary dentines. In situ hybridization has I Whilst the same fraction at isolated dentine ECM components allowed comparison of the expression of various transcripts in can initiate both reparative (Smith et al., 1990) and reactionary normal odontoblasts and odontoblast-like cells induced by various (Smith et al., 1994) dentinogenesis, it is probable that a synergistic growth factors (Begue-Kim et al., 1994). These studies have association of components is required to initiate the cascade of shown that odontoblasts and odontoblast-like cells, which are I

I Reactionary dentinogenesis 277

A E DENTINE

I EDTA

I O.15M eluted c:> heparin-agarose fraction ,I FM TO MP

--- o >f

Fig. 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 with the appearance of finger-like projections (arrows), x320. (D) Secretion of reactionary dentrne was limited to only those areas where the dentinal rubules were in communrcation with the cavity floor (arrows indicate outer limits of communication at cavity and pulp-dentine interface). x200. (E) Pulp-dentine interface beneath a control cavity lacking the implanted matnx fraction and showing no evidence of reactionary dentinogenesis, x200. (S-EI Black bars at bottom left of Figs. represents 10 microns). characteristic of primary and tertiary dentinogenesis, have the 3, BMP-2 and -4, bone sialoprotein and osteonectin, whereas potential for elaboration of similar matrix components. Normal either ubiquitous expression or no expression could be detected for odontoblasts and odontoblast-like cells induced by immobilized TGF-B2, IGF-1 or tibronectin mRNAs. Odontoblast-like cells ob- TGF-B1 or BMP-2 expressed transcripts encoding tor TGF-B1 and 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-B3 The nature of the molecules in the isolated dentine ECM transcripts. Thus, use of a panel of markers may be necessary to fractions which can interact with odontoblasts and stimulate characterize the phenotype at odontoblasts and odontoblast-like their activity may be varied, although involvement of growth cells. factors seems probable. The heparin-agarose bound material, The changes in activity of the odontoblast throughout its life and which stimulated reactionary dentinogenesis as described in the stimulation of its synthetic and secretory activity in response to the implantation experiments above contained significant appropriate stimuli resulting in reactionary dentine deposition amounts of TGF-B1, but not TGF-B2 or 3. Culture of dental indicate that the odontoblast is a very versatile cell. Morphological papillae with TGF-B1, or the closely related BMP-2, stimulated differences in the tubular structure of the matrix secreted by these extracellular matrix secretion by these cells (Begue-Kim et al., cells in primary, secondary and tertiary dentines could indicate 1992). This pattern at cell behavior was ditferent to that ob- molecular changes in these matrices. An increase in the secretion served when these growth factors were added with potentiating of extracellular matrix has been demonstrated in tertiary dentine molecules, such as heparin, when they were shown to be beneath carious lesions in human teeth (Lesot et al., 1993; Perry capable of inducing the ditferentiation of odontoblast-like cells and Smith, unpublished observations) and immunohistochemical (Begue-Kim et al., 1992). approaches have shown re-expression of collagen type III, There are many reports of the effects of TGF-B, and related fibronectin and the membrane 165 kDa fibronectin binding protein members at this growth factor tamily, on matrix synthesis. BMP-2 in this tissue (Magloire et al., 1988, 1992), although these three has been reported to influence the activity of cultured osteoblastic proteins have a transitory expression during the differentiation of MC3T3-E1 cells, where it stimulates alkaline phosphatase and primary odontoblasts (Lesot et al., 1990; Lukinmaa et al., 1991). collagen synthesis (Takuwa et al., 1991). TGF-B modulates the Differences in the expression of phosphoproteins in primary, synthesis of collagens (Ignotz and Massague, 1986; Yu et al., secondary and tertiary dentines have also been reported (Takagi 1991) and that at proteoglycans (Rapraeger, 1989; Yu et al.. 1991). and Sasaki, 1986; Aulak et al., 1991). It seems probable that these Clearly, TGF -B and related molecules can have a marked intluence various differences in matrix composition reflect modulation in on extracellular matrix synthesis, but it is important to note that, odontoblast activity. depending on its concentration, TGF-B can stimulate matrix syn- To investigate this further, the EDTA-soluble collagen, thesis orresorption (Tashjian et al., 1985; Hock et al., 1990). Thus, noncoliagenous protein and glycosaminoglycan levels have been the control mechanisms on the activity of these growth factors is of analyzed in reactionary/reparative dentines beneath carious le- paramount importance in determining their effects on cell behavior sions in human teeth (Perry and Smith, unpublished observations). in the tissue. The tertiary dentine specimens contained up to four times greater There are a number of levels at which control of growth factor levels of these components compared with primary dentine. How- activity could occur within the tissue. At the level of interaction with ever, these levels were not uniformly raised for the three classes of the cell, whilst considerable information exists on TGF-B receptors matrix components suggesting that there had been differential (Boyde et al., 1990) in general, little Information exists on these stimulation at the synthetic/secretory pathways for these compo- receptors in relation to odontoblasts. The presence ot the nents. Thus, it is clear that modulation of odontoblast activity can extracellular matrix proteoglycan, decorin, has been recently re- occur and that variation in expression of the different ECM compo- ported in dentine matrix (Lesot et al., 1994). Decorin not only binds nents can result. Attempts to relate these compositional changes TGF-B, but can also neutralize the activity of this growth tactor. The to the degree of regularity of the tissue tubular structure indicated time course for initiation of reactionary dentinogenesis when little relationship between these parameters. It is now important to isolated dentine ECM components are implanted in unexposed determinethe molecular basis otthis modulation of the odontoblasts cavities (Smith et al., 1994) suggests that the interactions of the and to investigate whether molecular stimuli can act on different active molecule(s) occurs on the cell body rather than the process aspects of the cell's synthetic/secretory processes. of the odontoblast. Further studies are now needed to elucidate the nature of the odontoblast growth factor interactions. Molecular basis of odontoblast stimulation in reaction- Control of growth factor activity can also occur by virtue of the ary dentinogenesis availability of the growth factor. Finkelman et al. (1990) have demonstrated the presence of a number of growth factors in Experiments on the implantation of isolated dentine ECM com- dentine, including TGF-B, and have suggested that the response ponents reported above (Smith et al., 1994) have demonstrated of odontoblasts to dental caries may be mediated by release of that interaction between these components and odontoblasts these growth factors from dentine during demineralization by results in stimulation of the synthetic/secretory activity of these bacterial acids. Our own studies have shown the presence of TGF- cells. Contact between odontoblasts and their extracellular matrix B1, 2 and 3 in dentine and that these molecules may be bound up appears to be important in the maintenance of their phenotype as in different extracellular matrix tissue compartments (Smith et al., well. The phenotypic morphology of these cells when cultured can unpublished observations). A proportion of these molecules can be only be preserved as long as contact is maintained between the released by demineralization, but some appear to be tightly asso- cells and dentine matrix (Munksgaard et al., 1978; Heywood and ciated with the collagenous matrix and require collagenase diges- Appleton, 1984). If the odontoblasts are removed from the tooth tion to release them. It is not clear yet as to whether these and cultured in isolation, they take on the appearance of a fibroblastic molecules are present in an active or latent state, but further morphology. It is possible to replace the mature dentine matrix by investigations are now required to understand the role of these preparations of isolated dentine ECM components as demon- molecules and whether they participate in physiological tissue strated by maintenance of odontoblast differentiation when dental function (perhaps helping to maintain the odontoblast phenotype) papilla were cultured in contact with such preparations for 4 days or if they are only available/exposed in pathological conditions (Lesot et al., 1986). when they can initiate a response of tertiary dentinogenesis. Rellc/jollar)' dell/jllogelll'sis 279

Conclusions JONTELl, M. and LINDE, A. (1983). Non-collagenous proteins of predentine from dentinogenically active bovine teeth. Biochem. J. 214: 769-776. Deposition of reactionary dentine by post-mitotic odontoblasts, KAWASAKI, K.. TANAKA, S. and ISHIKAWA. T. (1980). On the daily incremental lines in human dentine. Arch. Ora/Bioi. 24: 939- 943. which have ditterentiated during tooth development. can arise at lESOT, H., BEGUE.KIRN, C., KUBLEA, M.D., MEYER, J.M., SMITH, A,J., CASSIDY, the pulp-dentine interface and at a peritubular/intratubular location N. and RUCH, J.V. (1993). Experimental induction of odontoblast differentiation in response to an appropriate stimulus. Some of this reactionary and stimulation during reparative processes. Cells Marer. 3: 201-217. dentine may be a teature of normal physiological processes, such LESOT, H., KUBLER, M.D., FAUSSEA, J.L and RUCH. J.V. (1990). A 165 kDa as aging, although it is not clear as to what extent pathological membrane antigen mediating fibronectin.vincuhn interaclion is involved in murine processes such as dental disease, tooth wear etc. contribute to this odontoblast differentiation. Dlfferentiarlon 44: 25-35. "physiological" appearance. Probably the majority of reactionary LESOT, H.. SMITH. A.J., MEYER, J.M., STAUBLI, A. and RUCH, J.V. (1986). Cell- matrix interactions: influence of non-collagenous proteins from dentin on cultured dentine arises in response to dental disease. Traditional views of dental cells. J. Embryol. Exp. Morphol. 96: 195-209. the acid generated from plaque bacterial metabolism being the LESOT, H., SMITH, A_J., TZIAFAS. D., Bt:GUE-KIRN, C., CASSIDY, N. and RUCH, stimulus to the odontoblasts for reactionary dentinogenesis have J.V. (1994). Biologically active molecules and dental tissue repair: a comparative little evidence to supporfthem. It is likely that plaque bacterial acids review of reactionary and reparative denti nogenesis with the induction of odontoblast soiubilize growth factors, TGF-B in particular, as they permeate dlflerentiation in vitro. Cells Mater. 4: 199-218 dentine and that it is the solubilized growth factors that are LINDE, A. (1989). Dentin matrix proteins: composition and possible functions in calcificatIOn. Anat. Rec. 224: 154.166. responsible for initiating the stimulatory ettect on the odontoblasts LINDE, A. and GOLDBERG, M. (1993). Dentinogenesis. Crit. Rev. Oral Bioi. Med. 4: to secrete reactionary dentine. 679-728. LUKINMAA. Pol., MACKIE, E_J. and THESLEFF, I. (1991). Immunohistochemical Acknowfedgments 10ca1ization of the matrix glycoprotein tenascin and the ED-sequence containing The research was supported by the Commission of the European lorm of cellular fibronectin in human permanent teeth and periodontal ligaments. Communities SCIENCE Programme Contract no. SC1 ~-CT91-0680 J. Denl. Res. 70: 19-26. (WNSN). MacDOUGALL, M., ZEICHNER-DAVID, M. and SLAVKIN. H.C. (1985). Production and characterization of antibodies against murine dentin phosphoprotein. Biochem. References J. 232: 493-500. MAGLOIRE. H.. BOUVIER, M. and JOFFRE, A. (1992). Odontoblast response under AULAK, K., SMITH. A.J., MATTHEWS. J.B., POTTS. A.J.C.. PERRY, H. and carious lesions. Proc. Finn. Dent. Soc. 88 (Suppl. 1): 257-274. WilSON, C. (1991). Phosphoproteins 01dentine extracellular matrix: silver colloid MAGLOIAE. H.. JOFFRE A. and HARTMANN. D.J. (1988). Localization and synthe- staining. Biochem. Soc. Trans. 19: 188. sis of type III collagen and fibronectln in human reparative dentine. HistochemIstry BAUME, LJ. (1980). The biOlogy 01pulp and dentine. In Monographs in Oral Science, 88: 141-149. Vo1. 8 (Ed. H.M. Myers). S. Karger AG. Basel, pp. 67-182. MASSLER. M. and SCHOUR, I. (1946). The appositionalille span of the enamel and Bt:GUE-KIAN. C.. SMITH. A.J., LOR lOT. M.. KUPFEALE. C., AUCH, J.V. and dentin-forming celts. 1. Human deciduous teeth and first permanent molars. J. LESOT, H. (1994). Comparative analysis of TGF-f3s, BMPs,lGF, msxs, fibronectin. Dent. Res. 25: 145-150. and bone sialoprotein gene expressions during normal and in vitro induced MJOR. I.A. (Ed.) (1983). Denlin and pulp. In Reaction Patterns in Human Teeth. CRC odontoblasl differentiation. Int. J. Dev. Bioi. 38: 405-420. Press, Florida, pp. 63-156. BEGUE-KIRN, C., SMITH, A.J., RUCH, J.V., WOZNEY, J.M., PURCHIO, A., MJOR, IA and FURSETH, R. (1968). The inorganic phase of calcium hydroxide- and HARTMANN, D. and LESOT, H. (1992). Eflects 01 dentin proteins. transtorming corticosteroid-coveted dentin studied by electron microscopy. Arch. Oral Bioi. 13: growth factor 131 (TGFI31) and bone morphogenetic protein 2 (BMP2) on the 755.763. differentiation of odontoblast in vitro. 1m. J. Dev. Bioi. 36: 491-502. MUNKSGAARD, E.C., RICHARDSON. W.S. and BUTLER, W.T. (1978). Biosynthesis J_, BOYDE, F.T., CHEIFETZ, S., ANDRES, LAIHO, M. and MASSAGUE. J. (1990). of phOsphoprotein by rat incisor odontoblasts in in vitro culture. Arch. Oral Bioi. 23: Transtorming growth faclors 13-receptors and binding proteoglycans. J. Cell Sci. 583.585. 13 ($uppl.): 131-138. NAKAMURA. 0.. GOHDA, OZAWA, M., SENBA, I., MIYAZAKI. H., MURAKAMI. E" COUVE, E. (1986). Ultrastructural changes during the life cycle of human odontoblasts. T. and DAIKUHARA. Y. (1985).lmmunohistochemicat studies with a monoclonal Arch. Oral BioI. 31:643.651. antibody on the distribu1ion of phosphophoryn in predentin and dentin. CalClf. COX. C.F., WHITE. K.C., RAMUS, D.l., FARMER, J.B_ and SNUGGS. H.M. (1992). Tissue Int. 37: 491.500. Reparative dentine: factors affecting ils deposition. Ouintess. Int. 23: 257-270. PLANT, C.G., TOBIAS, R.S.. RIPPIN,J., BROOKS,JW. and BROWNE, R.M. (1991). FtNKELMAN. R.D., MOHAN. S., JENNINGS, J.C., TAYLOR, A.K., JEPSEN, S. and A study 01 the relationship among pulpal response, microbial microleakage and BAYLlNK, D.J. (1990). Quantitation of growth factors IGF-1, SGF/IGF-II and TGF- particle heterogeneity in a glass.ionomer base material. Dent. Mater. 7:217-224. 13in human denlin. J. Bone Miner. Res. 5: 717-723. RAHIMA, M., TSAY, T-G., ANDUJAR. M. and VEIS. A. (1988). Localization 01 FOX, A.G_ and HEElEY, J.D. (1980). Histological study of pulps 01 human primary phosphophoryn in rat inCisor dentin using immunocytochemical techniques. J. teeth. Arch. Oral Bioi. 25: 103-11 O. Hlstochem. CytOChem. 36: 153-157. FRANK, R.M., WOLFF, F. and GUTMANN, B. (1964). Microscopieelectroniquede la RAPRAEGER, A. (1989). Transforming growth factor (type 13)promotes the addition carie au niveau de la dentine humaine. Arch. Oral BiOI. 9: 163-179. of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia. J. Cell Bioi. 109: 2509-2518. GORTER DE VRIES. I., QUARTIER, E., VAN STEIRTEGHEM, A., BOUTE, P., COOMANS, D. andWISSE, E. (1986). CharachHLzationand immunocytochemical ROMAGNOLI. P., MANCINI, G., GAlEOTTI, F., FRANCHI, E. and PIEREONI, P. localization of dentine phosphoprotein in rat and bovine teeth. Arch. Oral Bioi. 31: (1990). The crown odontoblasts of rat molars horn pnmary dentinogenesis to 57-66. complete eruption. J. Denl. Res. 69: 1857.1862. HEYWOOD, B.A. and APPLETON, J. (1984). The ultrastructure of the rat incisor SCHOUR,I. and PONCHER, H.G. (1937). The rate of apposition of human enamel and odontoblast in culture. Arch. Oral Bioi. 29: 327-329. dentin as measured by the elfectsof acute fluorosis. Am. J. Dis. Child. 54:757-776. HOCK, J.M., CANALIS, E. and CENTRELLA, M. (1990). Translorming groWth factor- SELVIG. K.A. (1968). Effect 01fluoride on the acid solubility of human dentine. Arch. B slimulates bone matrix apposition and bone cell replication in cultured fetal rat Oral Bioi. 13: 1297- 1310. calvariae. Endocrinology 126: 421-426. SLOAN, A., CASSIDY, N., SMITH, A,J., Bt:GUE-KIRN. C.. RUCH. J.V. and LESOT, IGNOTZ, R.A. and MASSAGUE, J. (1986). Transforming growth factor. I) stimulates H. (1994). Culture of the dentine.pulp complex of rat incisor teeth. J. Dent. Res. the expression 01 libroneclin and collagen and their incorporation into the 73: 838. extracellu!ar matrix, J. Blof. Chern. 26J:4337-4345. SMITH, A.J., TOBIAS, A.S., CASSIDY, N., PLANT, C.G., BROWNE, R.M., BEGUE- JONES, S.J. and BOYDE, A. (1984). Ultrastructure of dentin and dentinogenesis. In KlAN.C., RUCH,J.v. and LESOT, H. (1994). Odonloblasl stimulation in ferrets Dentin and Dentinogenesis (Ed. A. Linde). CRC Press, Florida, pp. 81-134. by dentine matrix components. Arch. Oral Bioi. 39: 13-22.

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J.V. (1990). in vivo morphogenetic activity of dentine matrix proteins. J. 8iol. transforming growth taclors stimulate prostaglandin production and bone I Bucca/e 18: 123-129. resorption in cultured mouse calvaria. Proc. Nat!. Acad. Sci. USA 82: 4535- TAKAGI. Y. and SASAKI, S. (1986). HislOlogical dislribution of phosphophoryn in 4538. normal and pathological human dentins. J. Ora! Pathol. 15:463-467. TEN CATE, A.A. (1994). Oral Histology: Development. Structure and Function. Mosby, SI. Louis. TAKUWA, S. and NAGAI, N. (1971). Ultrastructure at rat odontoblasts in various I development and maturation. Arch. Oral Bioi. 16:993-1011. stages altheir TOBIAS, R.S" BROWNE. A.M.. PLANT,C.G., WILUAMS,J.A. and RIPPIN, J.W. TAKUWA. Y.. OHSE. C., WANG, EA, WOZNEY, J.M. and YAMASHITA, K. (1991). (1991). Pulpal response to two semihydrous glass ionomer luling cemenls. Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and Int. Endodont. J. 24: 95-1 07. collagen synthesis in cultured osteoblastic cells MC3T3. Biochem. Blophys. Res. YU. Y.M., BECVAR. R.oYAMADA, Y. and REDOI, A.H. (1991). Changes in the gene I Commun.174:96-101. expression of collagens, tibronectln.integrinand proteoglycansduringmatri)(- TASHJIAN, A.H., VOELKEL, E.F., LAZZARO, M., SINGER, F.R., ROBERTS, A.B., induced bone morphogenesis. Biochem. Biophys. Res. Commun. 177: 427-432.