Critical Reviews in Oral Biology & Medicine ... · 15(1):13-27 (2004) Crit Rev Oral Biol Med 13 Introduction Dentinogenesis has been studied extensively as investiga- tors attempt

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2004 15: 13CROBMMichel Goldberg and Anthony J. Smith

Cells and Extracellular Matrices of Dentin and Pulp: A Biological Basis for Repair and Tissue Engineering

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15(1):13-27 (2004) Crit Rev Oral Biol Med 13

Introduction

Dentinogenesis has been studied extensively as investiga-tors attempt to better understand the formation and min-

eralization of this collagenous connective tissue, which resem-bles bone but has its own distinct features. A unique feature ofdentin is that it is a mineralized tissue which surrounds thepulp, an unmineralized tissue, and gradually increases inthickness to the detriment of the space previously occupied bythe pulp. The dental pulp not only functions to provide nutri-tional and sensory properties to dentin, but also has its ownreparative capacity. This potential has important implicationsfor dental therapy.

The areas of odontoblast biology and dentinogenesis, pulpbiology, and the formation of reactionary and reparative dentinhave been extensively reviewed during the last two decades(Lesot et al., 1993; Linde and Goldberg, 1993; Ruch et al., 1995;Smith and Lesot, 2001). Together, these and many other excel-lent articles constitute the milestones of our present knowl-edge. However, despite this apparent wealth of information,many assumptions and questionable hypotheses still prevail.Clarification of many of these points is still required to under-pin future progress in our understanding of the properties ofpulp and dentin. For example, considerable data have beenobtained from studies on the early stages of dentin formationin embryonic tooth germs and simply extrapolated to the situ-ation in mature teeth. While there may be common features, thetissue architecture and cellular environments in tooth germs

and mature teeth are quite distinct. Following injury to themature tooth, pulp progenitor cells may be recruited during therepair processes and differentiate into second-generation odon-toblasts, neo-odontoblasts, or odontoblast-like cells. Thisplethora of names highlights our lack of understanding of theorigin, nature, and fate of these replacement cells. Do they arisefrom undifferentiated/stem cells (Mann et al., 1996; Gronthos etal., 2000) or from some other derivation? Answers to these andmany other questions may be key to our understanding of themechanisms responsible for repair in the tooth.

In this review, we will consider separately the biology ofdentin and pulp as a background to discussing the problems inpulp healing.

(I) Odontoblast Biology

(I-1) DERIVATION AND DIFFERENTIATIONOF ODONTOBLASTS

Odontoblasts are derived from the dorsal cranial neural crest(CNC). Early-migrating cranial midbrain crest-derived cellspopulate the dental mesenchyme of mandibular molar teeth inthe first branchial arch (Imai et al., 1996). Forebrain and mid-brain CNC-derived cells migrate to the fronto-nasal mass(Osumi-Yamashita et al., 1994). Non-CNC-derived mesenchy-mal cells are also present in the arch, and consequently in theregions where teeth develop. At the late bud stage, the con-densed dental mesenchyme consists of CNC-derived cells

CELLS AND EXTRACELLULAR MATRICES OF DENTINAND PULP: A BIOLOGICAL BASISFOR REPAIR AND TISSUE ENGINEERING

Michel Goldberg*

Faculté de Chirurgie Dentaire, Université Paris V-René Descartes, Groupe Matrices Extracellulaires et biominéralisations (EA 2496), 1, rue Maurice Arnoux, 92120 Montrouge, France; *correspond-ing author, [email protected]

Anthony J. Smith

University of Birmingham, School of Dentistry, St Chad's Queensway, Birmingham B4 6NN, UK

ABSTRACT: Odontoblasts produce most of the extracellular matrix (ECM) components found in dentin and implicated indentin mineralization. Major differences in the pulp ECM explain why pulp is normally a non-mineralized tissue. In vitro or invivo, some dentin ECM molecules act as crystal nucleators and contribute to crystal growth, whereas others are mineralizationinhibitors. After treatment of caries lesions of moderate progression, odontoblasts and cells from the sub-odontoblastic Höhl'slayer are implicated in the formation of reactionary dentin. Healing of deeper lesions in contact with the pulp results in the for-mation of reparative dentin by pulp cells. The response to direct pulp-capping with materials such as calcium hydroxide is theformation of a dentinal bridge, resulting from the recruitment and proliferation of undifferentiated cells, which may be eitherstem cells or dedifferentiated and transdifferentiated mature cells. Once differentiated, the cells synthesize a matrix that under-goes mineralization. Animal models have been used to test the capacity of potentially bioactive molecules to promote pulprepair following their implantation into the pulp. ECM molecules induce either the formation of dentinal bridges or large areasof mineralization in the coronal pulp. They may also stimulate the total closure of the pulp in the root canal. In conclusion, somemolecules found in dentin extracellular matrix may have potential in dental therapy as bioactive agents for pulp repair or tis-sue engineering.

Key words. Dentin, pulp, odontoblasts, bioactive molecules, tissue engineering.




together with an increasing number of non-CNC-derived cells.CNC-derived pre-odontoblasts are aligned at the periphery ofthe dental papilla and adjacent to the inner enamel epithelium(Chai et al., 2000). Consequently, many of the pulp cells may benon-CNC-derived cells.

Characterization of the signaling processes responsible forthe induction of odontoblast differentiation is fundamental tothe understanding of both physiological dentinogenesis as wellas tissue repair. The key roles played by growth factors in phys-iological odontoblast differentiation, and the recapitulation ofsuch events during repair, have recently been reviewed (Smithand Lesot, 2001). Considerable focus has been placed on thosegrowth factors, particularly of the Transforming GrowthFactor-beta (TGF-�) family, which may be directly involved insignaling cytodifferentiation of odontoblasts and odontoblast-like cells. Expression of growth factors by the inner enamelepithelium leads to their sequestration within the dental base-ment membrane for presentation to the pre-odontoblasts (Ruchet al., 1995). While proof of which growth factors are responsi-ble for signaling of odontoblast differentiation in vivo is lacking,Transforming Growth Factor �1 (TGF-�1), TGF-�3, BoneMorphogenetic Protein-2 (BMP-2), and Insulin-like Growthfactor-1 (IGF-1) (Bègue-Kirn et al., 1992, 1994) appear capable ofsignaling odontoblast differentiation in vitro.

Expression of growth factors by odontoblasts after differen-tiation may lead to their sequestration within the dentin matrix,where they may be released following injury to the tooth(reviewed by Tziafas et al., 2000; Smith and Lesot, 2001). Such apool of growth factors may be able to signal reparative process-es leading to odontoblast-like cell differentiation. Other growthfactors may be expressed by odontoblasts, but not sequestratedwithin the dentin matrix. For instance, TGF-�3 is expressedmore strongly than TGF-�1 (Sloan et al., 2000a) by human odon-toblasts, but is not detected in the dentin matrix. TGF-�1, butnot TGF-�3, has a high affinity for the proteoglycans decorinand biglycan within the dentin matrix, thereby facilitating itssequestration in this matrix. Thus, growth factors expressed byodontoblasts may play a variety of roles in tissue homeostasis,via either an autocrine or a paracrine action. Several angiogenicgrowth factors, presumably secreted by the odontoblasts

(Roberts-Clark and Smith, 2000), may be important in the vas-culature of the dentin-pulp complex, both physiologically andduring tissue repair. Caution will be required in studies of theexpression of growth factors in odontoblasts, since develop-mentally related temporal expression patterns may exist. Thus,the expression patterns in newly differentiated odontoblastswhen primary dentinogenesis is very active may not necessari-ly reflect patterns seen later in the life of the tooth.

A striking feature of the odontoblast is its post-mitoticnature. During tooth formation in the mouse, cells of the odon-toblastic lineage migrate from the central area of the dentalpapillae toward the periphery between days 14 and 18 ofembryogenesis. Mouse pre-odontoblasts multiply through 14 to15 cycles, with 10 hrs for each cell cycle (Ruch, 1987), althoughthe number of cycles and their length are often ignored inhumans. What is clear is that terminal differentiation requires aminimum number of cycles for cells to achieve competence torespond to inductive signaling (Ruch et al., 1976, 1982).

Following the last mitosis and prior to odontoblast differ-entiation, the pre-odontoblasts align perpendicular to the base-ment membrane (BM), and only the daughter cell adjacent tothe BM undergoes terminal differentiation into an odontoblast.This highlights the importance of the spatial configuration insignal presentation during odontoblast differentiation and theneed for this feature to be considered during any mimeticapproaches to pulp repair. It is assumed that the other daugh-ter cell, located away from the BM, becomes incorporated with-in the Höhl layer (Höhl, 1896), although direct evidence is lack-ing (Fig. 1).

(I-2) ULTRASTRUCTURE AND FUNCTIONAL

ACTIVITIES OF ODONTOBLASTS AND HÖHL CELLS

(I-2-1) Polarizing and young secretory odontoblasts

Young polarizing odontoblasts are small, ovoid cells with ahigh nucleus/cytoplasmic ratio, rudimentary rough endoplas-mic reticulum (RER), and a poorly developed Golgi apparatus.Essentially, they are cells undergoing re-organization as theydifferentiate in preparation for their secretory function. Withincreasing differentiation, gap-type junctions increase in num-ber and size between odontoblast cell bodies at locations wherethe distal junctional complex is actively developing. The distaljunctions form a physical barrier between the predentin com-partment and the odontoblast cell body.

Secretory odontoblasts are aligned along the periphery ofthe pulp on the formative surface of dentin, and both the pre-cise chronology of tooth formation and the ultrastructuralobservations of the odontoblast through its life-cycle suggestsome degree of pre-programming in the activity of these cells.Decreasing volume of the pulp chamber during secondarydentinogenesis has been reported to be associated with the pro-grammed cell death of odontoblasts. The nature of such pro-gramming of odontoblast activity or survival is unclear; how-ever, its elucidation could provide a powerful tool for the reg-ulation of dentinogenesis during repair. During tooth injuryleading to odontoblast death, the cells of the Höhl layer can re-express some transcription factors such as c-fos and differenti-ate into new odontoblasts for reparative dentinogenesis(Kitamura et al., 1999; Mitsiadis et al., 1999).

Functionally, the secretory odontoblast can be considered toconsist of two distinct parts: the cell body involved in the syn-

14 Crit Rev Oral Biol Med 15(1):13-27 (2004)

Figure 1. Rat molar. Polarized secretory odontoblasts (O) are seen inthe upper right. The sub-odontoblastic layer of Höhl cells (H) is con-tinuous with the outer part of the pulp (P). 650x.




thesis and control of cellular and extracellular proteins, and theprocess whereby secretion and limited re-internalization occur.The odontoblast process consists of one main trunk withnumerous lateral branches along its length. The process is limit-ed by a plasma membrane and contains predominantlycytoskeletal components. Actin microfilaments interconnectmicrotubules and are denser in the sub-plasmalemmal under-coat. Intermediary filaments of the vimentin type are also pre-sent. Nestin, another intermediary filament family member, ismainly found in neural-crest-derived cells, including odonto-blasts. In the main trunk, immunolabeling was positive fornestin, whereas small lateral branches were unstained. Bothcoated and uncoated small vesicles can be seen in the processes,and clathrin at the surface of membrane-coated vesicles hasbeen assumed to play a role in the internalization and backwardtransport of degraded or fragmented molecules from the ECM.Transport from the plasma membrane to the Golgi would allowfor turnover of ECM molecules and provide some retro-controlthrough stimulation or inhibition of ECM synthesis.

Lateral branching may contribute to cell-cell communica-tion and cell-matrix communication and may possibly beinvolved in peritubular dentin formation. Odontoblasts, likemany other cells, may use intermediary filaments to providespatial information. In mature dentin, there are minute open-ings of canaliculi into the lumen of the tubules. These canaliculicontain the lateral branches and constitute a tiny network thatinterconnects the tubules.

Compositional differences between predentin and the min-eralized dentin matrix led to the hypothesis that there may betwo levels of secretion from the odontoblast (Linde, 1984).Secretion of collagen and proteoglycans at the proximal levelwould give rise to predentin, while secretion of other non-col-lagenous components, including dentin phosphoprotein, at adistal level has been suggested to occur close to the mineral-ization front.

(I-2-2) Aged odontoblasts and apoptosis

Little is known about the ultrastructure of old odontoblasts,and much of our knowledge is based on the rat incisor, whichrepresents a rather special model for aging in view of its con-tinuous growth (Takuma and Nagai, 1971). While the compar-ison among polarizing, pre-secretory, young, and older secreto-ry odontoblasts is of course easier in a single tooth, it is unclearwhether such observations are relevant to teeth of limitedgrowth, as in humans. A reduction in length and cytoplasmicorganelles with aging would be in accord with a decreased syn-thetic and secretory capacity of these cells. An increase in num-ber and size of lysosomes and phagosomes would be expectedto be associated with gradual cellular degeneration and self-destruction. Whether this degenerative pattern is common toall teeth or is merely a reflection of the continuous growth ofthe rat incisor is unclear.

What is clear is that, with age, there is a reduction in thenumber of odontoblasts and pulp cells in both the rat (Murrayet al., 2002a) and the human (Murray et al., 2002b), and that thesecretory activity of these cells is reduced but may be re-acti-vated after injury. Upon re-activation, up-regulation of thefunctional activity of these cells would be expected to be asso-ciated with ultrastructural changes, but this information islacking. Observation of such changes is hampered by theabsence of chronological information on tissue changes duringinjury; consequently, it is often not easy to identify whether pri-

mary odontoblasts or a new generation of odontoblast-like cellsis being examined. The presence of tubular continuity amongprimary, secondary, and tertiary dentin, while not categoricalproof, is valuable in the identification of primary odontoblasts(Mjör, 1983).

The apparent longevity of odontoblasts and their capacityfor fluctuations in secretory activity, however, raise an interest-ing question. Are all odontoblasts the same, or are there differ-ences in the programmed life of the cells? Odontoblasts arepost-mitotic cells, but their numbers decline with age, and thereported apoptosis (Franquin et al., 1998) implies differences inthe life span of these cells. It is unclear if this is a programmedevent or whether it is the result of environmental influences,such as crowding during continued secondary dentin deposi-tion. Feedback from lysosomal accumulation of fragments ofdegraded molecules and/or specific gene expression mightalso possibly contribute to odontoblast survival. In view of thedevelopmental events leading to odontoblast differentiation, itis hard to envisage programmed differences in cell life span.Despite differences in survival time between odontoblasts,their longevity compared with that of many cells of the body isremarkable and suggests a strong influence of expression of cellsurvival factors.

(1-3) THE EXTRACELLULAR MATRIXOF PREDENTIN AND DENTIN

Varieties of molecules have been identified inpredentin/dentin, and more will almost certainly be reported.They have been extensively reviewed by many authors (forreview, see Butler et al., 2003), and we shall focus here only onthe comparison between dentin and pulp matrix components,in relation to their properties to promote mineralization andrepair of the tissues. The Table summarizes our present knowl-edge.

The non-collagenous proteins may be subdivided intomolecules predominantly found in mineralized tissues (boneand dentin), and more ubiquitous molecules. For many years,some proteins, such as dentin phosphoprotein (DPP) anddentin sialoprotein (DSP), were believed to be present exclu-sively in dentin (MacDougall et al., 1997) It is now establishedthat bone cells also synthesize DSP, and probably DPP, but in aratio that is estimated to be about 1:400 of that in dentin (Qin etal., 2002). Dentin matrix protein-1 (DMP-1) is expressed by bothbone and dentin (George et al., 1993). The gene encoding thisprotein is found near the locus of the DSPP gene. This is alsothe locus where the dentin genetic defect dentinogenesisimperfecta type II has been identified. In vitro studies haveshown that these phosphorylated proteins can be nucleators,and they have been strongly implicated in crystal growth dur-ing dentinogenesis, although the nucleating characteristics ofthese molecules in vivo remain to be established. In addition toa role in dentin mineralization, these molecules exhibit somesignaling function, adhesive properties through their RGDsequence, and may possibly be protective against complement-mediated cell toxicity. Recently, 4 molecules have been groupedas a family of Small Integrin-Binding Ligand N-linkedGlycoproteins (SIBLINGs) (Fisher and Fedarko, 2003). Theseinclude DSPP, and consequently DPP and DSP, DMP-1, bonesialoprotein (BSP), and osteopontin (OPN). BSP and OPN arefound mostly in bone but are also present in odontoblasts anddentin. The Matrix Extracellular Phosphorylated protein(MEPE) is a more distantly related member of the SIBLING

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family, as is also the case for enamelin.DPP and DSP have long been considered as unique phe-

notypic markers of dentin and secretory odontoblasts. Sincebone cells also expresses DSPP, DSP, and DPP, these can hardlybe considered as dentin-specific molecules, as is also the casefor DMP-1. Pre-secretory ameloblasts also express these mole-cules at some very early stages of odontogenesis. Thus, the listof the various molecules presented in the Table and the lack ofabsolute specificity for any of them suggest that, for the identi-fication of cells presumed to be of the odontoblastic lineage, apanel of various markers must be considered. The combinationof expression of DPP, DSP, DMP-1, and nestin may be valuable.However, we still have very limited understanding of how the

expression of these variouscomponents is modulatedunder different conditions.

Some molecules that arenot phosphorylated havebeen identified in dentin. The�-carboxyglutamic-contain-ing molecule osteocalcin,present in odontoblasts, waslong believed to be associatedwith mineralization. How-ever, increased mineralizationwas observed in bones ofmice showing deletion of thisgene (Dulcy et al., 1996), thusrequiring revision of the roleof this protein.

The presence of amelo-genin in dentin was firstattributed to a degradationprocess. It was hypothesized,and partially demonstratedimmunohistochemically, thatamelogenin migratesthroughout dentin and is fur-ther incorporated (internal-ization) into odontoblastprocesses and pulp cells(Nakamura et al., 1994).Enamelysin (MMP20), whichacts on amelogenin as sub-strate, has been detectedwithin odontoblasts andlends support to the hypothe-sis of active degradation ofamelogenin following inter-nalization. However, it wasrecently demonstrated thatalternatively spliced amelo-genins, expressed either by allexons (A+4) or by all exonsexcept exon 4 (A-4), are alsosynthesized by odontoblastsand may be isolated frombovine and rat dentin. In pre-liminary studies, A+4 and -4were identified as a differenti-ation factor and a chondro-cyte-inducing agent (CIA).

They interact with bone and cartilage transcription factors(Nebgen et al., 1999; Veis et al., 2000).

Other growth factors have been identified in dentin,including TGF-�, IGF-1 and -2, FGF-2 (Finkelman et al., 1990;Cassidy et al., 1997; Zhao et al., 2000), and various angiogenicgrowth factors (Roberts-Clark and Smith, 2000). The state ofactivation of some of these growth factors is unclear, althoughmuch of the TGF-� appears to be in active form and to be asso-ciated with various ECM components of dentin (Smith et al.,1998). This is corroborated by the biological effects of dentinmatrix preparations containing TGF-�1 on odontoblast differ-entiation and the inhibition of these effects by neutralizing anti-bodies (Bègue-Kirn et al., 1992). However, in mice overexpress-


TABLECells and Extracellular Matrix Components (ECM) Found in Dentin and Pulp

Dentin Pulp

Cells Odontoblasts exclusively Fibroblasts (pulpoblasts), vascular cells,pericytes, neural cells, histiocytes/ macrophages, dendritic cells, lymphocytes, mast cells

Collagens Types I and I trimer (98%) Type I (56%)Types III (1-2%) and V (1%) Types III (41%) and V (2%); Type VI (90% of the dentin ECM) (0.5%) associated with microfibrillin

Non-collagenous (10% of the dentin ECM)proteins

Phosphorylated matrix proteins (SIBLINGs):DSPP > DSP and DPP noneDMP-1, BSP, OPN, MEPE BSP, OPN

Non-phosphorylated matrix proteins: FibronectinMatrix GLA protein, osteocalcin, osteonectin Osteonectin (in tooth germs)

Proteoglycans (SLRPs) VersicanCS/DS PGs: decorin-biglycan(CS-4 81%, CS-6 14%, CS/DS 2%) CS-4 and -6, 60%; DS, 34%; KS, 2%KS PGs: lumican, fibromodulin, osteoadherin Hyaluronic acid

Amelogenin 5-7 kDaGrowth factors: TGF-�, ILGF-I and -II, BMPsFGF-2, VEGF, PDGF Types IA and II receptors for TGF-�,

activin, and BMPs

Metalloproteinases: collagenase (MMP-1), MMPs: collagenases, gelatinases (MMP-2 and -9), gelatinases, stromelysin-1stromelysin-1 (MMP-3), enamelysin TIMPs(MMP-20), MT1-MMP, TIMP-1 to -3

Alkaline phosphataseSerum-derived proteins:�HS2-glycoprotein, albumin, lipoproteins Fibronectin of serum origin

Phospholipids:Membrane phospholipids (66%) Membrane and ECM phospholipidsExtracellular-mineral-associated phospholipids (33%)




ing TGF-�1, reduced expression of DSPP was detected and ledto defective hypomineralized dentin formation (Thyagarajan etal., 2001). Interaction with ECM components of the dentinmatrix may confer protective effects on growth factor activity,since activated TGF-�1 at least has a relatively short half-life.The presence of these growth factors in dentin emphasizes thebiologically active nature of the matrix and the need to moveaway from regarding it as a relatively inert material.

The diverse range of molecules found in dentin has chal-lenged researchers for several decades and still poses numer-ous questions in terms of their functional relationships. Ourunderstanding of their precise tissue locations is still limited,and yet this is fundamental to an appreciation of their func-tions. The complex interactions between and among many ofthese molecules may be critical to their function in tissue devel-opment and homeostasis, and our appreciation of such interac-tions is only starting to unfold. Nevertheless, it is clear that sev-eral of these molecules may have potent biological effects in thereparative situation, and that the matrix interactions occurringmay be important regulators of reparative processes.

(I-4) DENTIN

Dentin is a complex heterogeneous structure, a manifestationof the cellular processes that take place during its formation.Thus, mantle dentin reflects secretory events that occur as theodontoblasts complete their differentiation. The main bulk ofcircumpulpal dentin is comprised of inter- and peri-tubulardentin matrices permeated by dentinal tubules containing theodontoblast processes as these cells retreat in a pulpal direc-tion. Estimates of tubular density have varied from 18,000 to83,000 per square millimeter, but a mean of 21,000 in deeperareas and 18,800 in the middle areas of dentin appears morerealistic (Schilke et al., 2000).

During phylogenetic development, osteodentin, which isfound in ancestral species, disappears, and orthodentinbecomes predominant. Matrix of osteodentin morphology isstill detected sometimes in pathological dentin, e.g., duringgenetic diseases, drug-induced diseases, or in response tocaries. The persistent presence of odontoblasts on the formativesurface of orthodentin has implications for their capacity to beup-regulated and participate in repair by secreting tertiarydentin (Smith et al., 1995), thereby allowing for the depositionof dentin at the pulp/dentin interface. In contrast, osteocyte-like cells become entrapped within the osteodentin matrix, thuslimiting their capacity to contribute to reparative processes.

(II) Pulp Biology

(II-1) THE PULP DURING CROWNAND ROOT FORMATION

Most tooth germs develop within an environment derivedfrom the first branchial arch with the exception of the maxillaryincisors, which originate from the fronto-nasal bud. The futurepulp contains a mixture of resident mesenchymal cells, alreadypresent at the site where the dental lamina and tooth budsdevelop, together with para-axial mesenchymal-derived cellsand cells migrating from the cranial neural crest (CNC).

It is generally assumed that the derivation of the differentpulp cell populations in the crown and root of the tooth is sim-ilar, but evidence to support this view is lacking. The epithelial-mesenchymal interactions responsible for tooth development

in the crown and root and culminating in odontoblast differen-tiation show many parallels, although differences do exist inthe structure of the epithelial tissues. For example,ameloblastin (also named amelin or sheathlin) and enamelin,but not amelogenin, are expressed in the Hertwig root sheath(Bosshardt and Nanci, 1997). This is in contrast to the situationin the enamel organ during crown development, where amelo-genin contributes 90% of the secreted matrix.

The morphogenesis of the vasculature is quite different inthe pulp of the crown and root. In the crown, blood vessels thatpenetrate the apical area of the pulp infiltrate and brancharound the odontoblast layer. As seen in corrosion resin casts ofdog pulp, the sub- and odontoblastic layers are fed by capillar-ies forming successive small glomerular well-individualizedstructures that feed limited areas about 100-150 �m wide. Incontrast, in the root, resin casts indicate a somewhat continu-ous network of sub-odontoblastic capillaries (Takahashi, 1985).In the root, arteries and veins are also located centrally,although the vessels in the two parts of the pulp appear quitedistinct as a result of their formation at different stages of toothdevelopment.

Crown and root differences in nerve development alsoexist, although information on events in the root is limited. Thenumber of nerve fibers does not appear to be stable in thetooth, perhaps reflecting the changing environment to which itis subjected. This implies a capacity for neural regenerationwithin the pulp and offers exciting opportunities for exploita-tion in any approaches to the regeneration of pulp tissue.

Differences in development of the crown and root of thetooth have received only limited attention and yet may holdvaluable clues to differences in tissue structure and behavior inthese two regions of the tooth. In the absence of more definitiveinformation, caution is required in assumptions of similaritiesbetween the crown and root of the tooth.

(II-2) THE PULP IN THE MATURE ADULT TOOTH

(II-2-1) Pulp cells

Once tooth formation is complete, the pulp is totally surround-ed by a mineralized environment, and because of the continu-ous slow secretion of physiological secondary dentin, the spaceoccupied by the pulp is gradually reduced. There is still, how-ever, communication via the apical foramen between the pulpand the periapical tissues.

The adult pulp contains cells that are responsible for theformation and turnover of a complex non-mineralized ECM.Most of the cells are fibroblast-like cells (Fig. 1). Macrophages,nerves, and capillaries may also be observed. Division of thesecells within the crown of the adult pulp appears to be limited,although cell renewal following apoptosis probably occurs.While, superficially, all the fibroblasts appear morphologicallysimilar, variations in their proliferative activity suggest thatthey represent a heterogeneous cell population (Moule et al.,1995). Such heterogeneity is further suggested from studies inhuman deciduous teeth where 183 fibroblast strains were iso-lated, with only 6 of these strains being capable of forming min-eralization nodules (Tsukamoto et al., 1992). These observationsindicate that isolation of primary cultures of pulp cells maygive rise to considerable selection of cell phenotype, which inturn could influence the interpretation of data from these cells.

Pulp fibroblasts are elongated, with a large nucleus and




well-developed RER. The Golgi apparatus located near thenucleus and the presence of secretory vesicles reflect the syn-thetic capacity of these cells. Lysosomes are also present,although it is not known if the same fibroblasts are involvedsimultaneously in the synthesis and catabolism of ECM com-ponents. Nevertheless, there appears to be considerableturnover of ECM components, particularly in the youngerpulp. Pulp fibroblasts do not exist in isolation, but are connect-ed by desmosome-like and gap junctions, which facilitate inter-cellular communication. This also implies that if the cells moveor translocate, this communication may be broken. This motil-ity is perhaps facilitated by the presence of �-smooth-muscleactin, which contributes to contraction of the cells and/or theextracellular matrix (Alliot-Licht et al., 2001).

Defense cells have also been identified in the healthy pulp,including mostly dendritic cells and histiocytes/macrophages.A few lymphocytes and mast cells are also present. Most, if notall, are Class II molecule-expressing cells, implicated inimmune responses. Some years ago, Jontell et al. (1987) report-ed the presence of immunocompetent cells in normal pulp.Cells expressing the major histocompatibility complex (MHC)class II molecules and with characteristic dendritic morpholo-gy have been identified in the pulp (Jontell et al., 1998). B-lym-phocytes are rarely encountered in the normal dental pulp, butmacrophages are present, probably for phagocytic activity.These various cells are especially numerous in the peripheralareas of the pulp, where they may participate in the immuno-surveillance of this tissue and contribute to the pulp responsesto dentinal caries (Jontell et al., 1998; Okiji et al., 2002).Cooperation between and among the various cell populationsin the pulp may be an essential feature of normal tissue homeo-stasis, including pulp cell dynamics, and such interactionsmust be elucidated if we are to understand the physiologicalbehavior of pulp and its response to injury. While the media-tion of such interactions is poorly understood at present, inter-esting insights have arisen from null mutation studies for thegrowth factor TGF-�1 (D'Souza et al., 1998). This growth factor,which is physiologically expressed by pulp cells together withits other isoforms (Sloan et al., 2000a,b), is a potent immuno-suppressant and may play a key role in the regulation ofinflammatory cell activity in the pulp.

(II-2-2) Pulp extracellular matrix

Pulp fibroblasts produce a complex ECM, which is substantial-ly different from that of dentin and other soft connective tissues(see Table). In the pulp, large intercellular spaces contain type Iand type III collagen fibrils (56 and 41%, respectively), in con-trast to the predominantly type I collagen of dentin. Types V(2%) and VI collagen (0.5%) are also found but in lesseramounts. Microfibrils 10-14 nm in diameter containing fibrillinare associated with microfibrils of type VI collagen. Differencesin the non-collagenous ECM components also exist betweenpulp and dentin. Fibronectin, of both pulp and serum origin, isprevalent in pulp. Chondroitin 4- and 6-sulfate (60%), der-matan sulfate (34%), and keratan sulfate (2%) glycosaminogly-cans (GAGs) are associated with protein cores and, conse-quently, are found as proteoglycans (PGs) in the pulp. AmongPGs, decorin, biglycan, and versican are present. This patternof PGs in pulp differs substantially from that in dentin, whereCS-4 accounts for 81%, CS-6 for 14%, and CS/DS for 2%.Together with hyaluronic acid, which accounts for about 2% ofthe GAGs found in the pulp, pulp PGs contribute significantly

to the viscosity of the intercellular matrix of the pulp. Major dif-ferences also exist in the lipids found in pulp and dentin(Goldberg and Septier, 2002).

While the derivation of the pulp cells may be key to ourunderstanding of their secretory activity, it is probable that con-trol of their behavior by growth factors and cytokines will alsobe important. Bone Morphogenetic Proteins (BMPs), their tran-scripts, and the types IA and II receptors are expressed by pulpcells (Takeda et al., 1994a,b; Gu et al., 1996). Moreover, pulp cellsexpress many members of the TGF-� superfamily, types I andII receptors for TGF-�s, activins, and BMPs (Toyono et al., 1997;Sloan et al., 1999, 2000a). The effects of the TGF-� family mem-bers on pulp cell behavior are poorly understood, but couldprove a fruitful area of study, especially with respect to pulpmineralization.

The lack of mineralization in pulp might be explained bythe absence of specific molecules which have been identified indentin. Immunostaining and in situ hybridization suggest thatDSP, DPP, DMP-1, and osteocalcin are predominantlyexpressed by odontoblasts. Osteonectin, found widely in thedental papilla of tooth germs, is found in the odontoblastsalone, and not in the pulp in adult teeth. Osteopontin and BSPare present in both dentin and pulp (Yokota et al., 1992). Thesesubstantial differences in composition between dentin andpulp could partly explain why pulp is unable to mineralizeunder physiological conditions. The presence of mineralizationinhibitors (e.g., the high concentration of PGs) in pulp may alsocontribute to the absence of mineralization. Thus, the regula-tion rather than the initiation of mineralization may be themore crucial question to be addressed, i.e., what prevents min-eralization in pulp and other soft connective tissues rather thanwhat initiates it in dentin.

(II-3) RENEWAL OF CELL POPULATIONSIN NORMAL AND EXPERIMENTAL CONDITIONS

Little is known of the life span of pulp fibroblasts. A decline inthe total number of cells has been reported in both the humanand rat pulp with aging (Lavelle, 1968; Bernick, 1987). Only lim-ited inferences can be drawn from these studies, since noattempt was made to distinguish among different pulp cell pop-ulations. Significant decreases in numbers of sub-odontoblastcells with aging have been observed in the human and rat pulp(Murray et al., 2002a,b). Interestingly, with increasing age, whilethe fibroblast density decreased in the human pulp, small butsignificant increases in fibroblast density were seen in the rat.While these observations relate to numbers rather than turnoverof pulp cells, analysis of the data suggests that perhaps fibro-blast density is less-well-regulated than that of other cells of thepulp in the rat, and also than fibroblasts of the human pulp.

Significant increases in cell numbers were seen in the cen-tral pulp core and within the peripheral sub-odontoblast layerafter administration of a fatty-acid-deficient diet (Vermelin etal., 1995). These workers hypothesized that killer cells are nor-mally responsible for destruction of excess pulp cells to main-tain a balance in cell numbers, and that their action wasimpaired by this diet, thereby disrupting the normal dynamicsof pulp cell renewal. Such increases in cell density under exper-imental conditions are in contrast to the low rate of cell divisionnormally seen in the coronal pulp. This may indicate that therate of cell division can fluctuate with the experimental condi-tions or that existing approaches to the study of pulp cell divi-sion have not been capable of identifying all dividing cells. [3H]





thymidine studies of pulp cell division beneath exposed cavi-ties restored with calcium hydroxide to investigate odonto-blast-like cell differentiation suggested that dividing cells movefrom the central to peripheral sites of the pulp (Fitzgerald et al.,1990). The existence of a true self-renewing pulp stem cell pop-ulation (Gronthos et al., 2000, 2002; Young et al., 2002) hasimportant implications for odontoblast-like cell differentiationduring repair and is discussed further below. Our knowledgeof pulp cell dynamics is still limited, and yet the renewal andpotentiality of different cell populations in the pulp are key tounderstanding and exploiting the behavior of this tissue.

Cell death among the different cell populations in the pulpis still poorly understood. Using the TUNEL procedure todetect apoptosis in rat molars, investigators have detected afew positive cells in the outer areas of pulp (Vermelin et al.,1996). Because of the false-positive results that may be seenwith the TUNEL method, other approaches have also beenadopted. Immunostaining of healthy human pre-molars fortransglutaminase revealed restriction of positive cells, again, tothe outer areas of the pulp, and, ultrastructurally, apoptoticcells in young rat molar pulps show characteristic morpholog-ical changes (Vermelin et al., 1996). In this context, it has beensuggested that apoptotic bodies may contribute to the initiationof biological mineralization (Kardos and Hubbard, 1982). Thus,the role of apoptosis may go beyond the simple regulation ofcell density and renewal of cell populations.

(III) Pulp Repair

(III-1) REACTION OF DENTIN AND PULP TO MILDINJURY—HEALING, REGENERATION AND REPAIR

(III-1-1) Reactionary dentin

The ability of the odontoblast to respond to injury (e.g., caries,cavity preparation) and up-regulate its secretory activity lead-ing to deposition of reactionary dentin is well-established(Smith et al., 1994). The important feature of this response isthat there is no cell renewal and the odontoblasts have to sur-vive the injury. This is in contrast to reparative dentinogene-sis, where the intensity of the injury is of a magnitude thatresults in odontoblast death and cell renewal by a new gener-ation of odontoblast-like cells (Lesot et al., 1993; Smith et al.,1994). The process of reactionary dentinogenesis involves up-regulation of odontoblast activity, often in quiescent cells atthe stage of physiological secondary dentinogenesis, inresponse to the injury stimulus. The nature of the signalingprocess from this stimulus may be rather variable and hasbeen hypothesized to result from the release of growth factorsand other bio-active molecules from the dentin matrix duringinjury (Smith et al., 1995). Consequently, the up-regulatorysignaling may be rather non-physiological and lead to com-positional differences in matrix secretion during primarydentinogenesis. Behind a calcio-traumatic line (Fig. 2), a moreor less tubular dentin is secreted, bearing most of the charac-teristics of orthodentin, although not always positivelystained by the "Stains-all" method. This suggests that reac-tionary dentin does not always contain phosphorylated pro-teins (Takagi and Sasaki, 1986), or perhaps that the proteinspresent at that location are not phosphorylated. By definition,reactionary dentin is secreted by surviving odontoblasts, andthus other pulp cells are not involved in its synthesis. A vari-

ety of bioactive molecules may participate in the signaling ofreactionary dentinogenesis, although relatively few have beencharacterized. Members of the TGF-� family, including TGF-�1, TGF-�3, and BMP-7, are capable of up-regulation of secre-tion (Sloan and Smith, 1999; Sloan et al., 2000b), althoughundoubtedly there may be other molecules that are capable ofparticipation in these signaling processes.

The injury to the odontoblast that produces a reactionarydentinogenic response may well be responsible for the calcio-traumatic line that delineates the dentin matrix secreted pre-and post-injury. This line is indicative of an aberration in matrixsecretion and mineralization and implies that the injury result-ed in abnormal odontoblast behavior at this point. The rela-tionship between the degree of injury that an odontoblast canwithstand and still survive is unclear. Correlation of carieslesion progression and reactionary/reparative dentinogenicevents is hampered by lack of chronological information on tis-sue changes that would distinguish odontoblast survival andrenewal. Morphological changes in odontoblasts beneath carieslesions have been reported (Bjørndahl et al., 1998), and in veryactive lesions, tertiary dentinogenic processes may be absentaltogether (Bjørndahl and Darvann, 1999). However, these datado not allow for discrimination of reactionary from reparativedentinogenesis. Similar problems can exist in the study of pul-pal responses beneath cavity preparations where surgical pro-cedures can cause odontoblast death (Kitamura et al., 2001).Nevertheless, examination of the relationship among depth ofcavity preparation, odontoblast numbers, and the tertiarydentinogenic response beneath the cavity indicates that if thecavity is prepared carefully enough, extensive odontoblast lossis seen only when pulpal exposure is approached (About et al.,2001; Murray et al., 2001). This raises an important question asto the ability of the odontoblast process to self-repair after cav-ity preparation. The latter studies imply that it is only when theprocess is cut near the cell body that the odontoblast is nolonger able to self-repair such injury, since it is unlikely that anycell could withstand an on-going loss of integrity in its mem-brane. The nature of the repair events that might result in resti-tution of the integrity of the membrane of the cut odontoblastprocess, however, remains to be elucidated and will provide afruitful area of study.


Figure 2. Human tooth. Reactionary dentin is delineated from thedentin by a calcio-traumatic line (arrowheads) and from the pulp bypredentin and odontoblast layers. 360x.




(III-1-2) Reparative dentin and odontoblast-like cells—factors influencing the recruitment and differentiationof secondary odontoblasts

With injury to the tooth of greater or more sustained intensity,localized odontoblast death will probably result. If suitable tis-sue conditions prevail, a new generation of odontoblast-likecells or "secondary odontoblasts" may differentiate from pro-genitor cells within the pulp and secrete a reparative dentinmatrix. In the case of the injury leading to pulpal exposure, thisreparative dentinogenesis may give rise to dentin bridge forma-tion. The different possible derivations for the progenitor cellsthat have been suggested to give rise to this new generation ofodontoblast-like cells have been alluded to above (Section II-3).They include the sub-odontoblast cells in the layer of Höhl,fibroblasts, undifferentiated mesenchymal cells from the pulpcore, and vascular-derived pericytes. It is unclear, however,whether any of the adult-derived resident progenitor cell popu-lations represents a defined stem cell population (Gronthos etal., 2000, 2002; Young et al., 2002). Dedifferentiation and trans-differentiation of certain mature cells lead to re-evaluation of theconcept of stem cells in repair processes.

It is possible that all of these derivations for the progenitorcells are valid and that the term "odontoblast-like cell" has beenused rather loosely to describe any cell in the pulp capable ofdepositing a mineralized matrix after injury to the tooth. Thephenotype of the primary odontoblast may be defined by themorphology of the cell and the matrix it secretes as well as by itspattern of gene expression, leading to the synthesis and secre-tion of characteristic dentin matrix proteins. Few odontoblast-like cells would fulfill all of these criteria, although it must berecognized that they are cells capable of repair and thereforemay display non-physiological traits. Nevertheless, it seemslikely that, at times, the term "odontoblast-like cell" may havebeen used inappropriately. Attempts should be made to relatethe cell originating from the progenitor cell to the phenotype ofthe cell responsible for repair in any pulpal injury situation.

The spectrum of matrix appearance observed during pulprepair can range from an atubular fibrodentin to a relativelyregular tubular reparative dentin. A fibrodentin response mayoften precede secretion of a more tubular dentin on its surface,which raises questions as to the dentinogenic specificity of theformer. Interpretation of many of these tissue changes is oftenhampered by the absence of data either on gene expression bythe cells responsible for the matrix secretion or on the nature ofthe matrix components secreted. While attempts have beenmade in some studies to characterize the cell phenotype duringin vivo experimental reparative dentinogenesis (D'Souza et al.,1995), in vitro studies may provide a simpler model for inter-pretive purposes (About et al., 2000).

A striking feature of the comparison between primaryodontoblast and odontoblast-like cell differentiation is theabsence of involvement of dental epithelium in the latter situa-tion. The inductive signal for odontoblast differentiation dur-ing tooth development has been proposed to be growth factorin nature, probably of the TGF-� family (Smith and Lesot,2001). TGF-� isoform expression by odontoblasts (Sloan et al.,2000b) leads to their sequestration within the dentin matrix(Cassidy et al., 1997), from where they may be released (Smithet al., 2000) or exposed (Zhao et al., 2000) by demineralizingagents. Dentin matrix extracts are capable of inducing odonto-blast-like cell differentiation when implanted in vivo within

exposed cavity preparations (Smith et al., 1990). Inclusion ofTGF-� neutralizing antibodies blocked odontoblast-like celldifferentiation (Bègue-Kirn et al., 1992). While TGF-�1, TGF-�3,follistatin, BMP-2 or -4, and IGF-1 all showed some effects onodontoblast-like cell differentiation in cultured dental papillae,only TGF-�s stimulated gradients of odontoblast-like function-al differentiation over large areas (Bègue-Kirn et al., 1994). Invivo implantation of TGF-�1 or BMP-2, -4, or -7 in pulp-cappingsituations (Rutherford et al., 1993, 1994; Nakashima, 1994a,b;Nakashima et al., 1994; Six et al., 2002b) and implantation atcentral pulpal sites (Tziafas et al., 1998) gave rise to variableresults. The complex injury situation modeled in these studiesmay have been influenced by the mode of presentation of thegrowth factor for signaling cell differentiation and could havecontributed to heterogeneous data. However, in a simplermodel of injury involving a needle-punch injury to the odonto-blast layer in cultured tooth slices, TGF-�3, but not TGF-�1,was capable of inducing odontoblast-like cell differentiation inassociation with the agarose beads used for growth factordelivery (Sloan and Smith, 1999). De novo reparative dentino-genesis involving odontoblast-like cell differentiation could beobserved when TGF-�1, in an alginate hydrogel, was appliedto the cut pulpal surfaces of cultured human tooth slices (Dobieet al., 2002). Clearly, anomalies exist in the reported effects ofthe different members of the TGF-� family, and especially TGF-�1 and -�3, on odontoblast-like cell differentiation. Differencesin effects may simply reflect immobilization of the growth fac-tor at its site of action by interaction with the extracellularmatrix components (Smith et al., 1998), or there may be arequirement for presentation of the growth factor in a particu-lar conformational or complex arrangement. Detailed studiesof signal transduction events during odontoblast-like cell dif-ferentiation are required to correlate the nature of signalingprocesses with specific morphological and functional changesin the cell.

(III-1-3) Odontoblast-like cells: gene regulation and transcription factors

Little is known of gene regulation and transcription factors thatare involved in the recruitment and differentiation of odonto-blast-like cells. However, those approaches provide powerfultools to aid our understanding of the cellular events takingplace.

Tooth cavity preparation models of dental injury have beenwidely used to study odontoblast-like cell differentiation eitherwith or without mechanical exposure of the pulp. Under care-fully controlled conditions, cavity preparation can be undertak-en without death of the underlying odontoblasts (Smith et al.,1994). However, odontoblast death can be readily induced dur-ing cavity preparation, even to the extent of aspiration of odon-toblast nuclei within the dentinal tubules (Mjör, 1983). Animalmodels with smaller teeth may be more susceptible to injuryduring cavity preparation than larger teeth, where dissipationof the heat generated by cutting is easier. The cellular responsesto these various degrees of injury are likely to be quite wide-ranging and should be considered in the comparison of datafrom different studies. Molecular characterization of the cellularresponses to injury may be valuable in assessments of its impacton repair. Apoptosis was observed in odontoblasts 1 hr aftercavity preparation in rat molars and was seen after 1 day in theunderlying cells of the sub-odontoblastic region, with parallelexpression of Bcl-2 (Kitamura et al., 2001).





A variety of molecules probably contributes to the signal-ing cascade, resulting in odontoblast-like cell differentiation.The nuclear proto-oncogenes, c-jun and jun-B, are known tocontrol transcription via a factor termed "activator protein-1"(AP-1), which is stimulated by growth factors such as BMPs,Tumor Necrosis Factor, and Insulin-like Growth Factor. AP-1stimulates the transcription of osteocalcin, alkaline phos-phatase, and collagens. From 3 to 7 days after cavity prepara-tion, c-jun and jun-B are expressed in the pulp cells underlyingthe cavity. After 14 days, they are expressed only in the forma-tive cells lining the reparative dentin layer (Kitamura et al.,1999). Notch receptors and the Delta 1 ligand are expressedduring tooth development, but not in adult teeth except afterinjury, when they appear to be up-regulated again. Notch 3 isassociated with vascular structures, whereas Notch 1 is mostlyfound in a few pulp cells close to the injury site (Mitsiadis et al.,1999). Pulp cells involved in reparative dentinogenesis havebeen reported to express collagen type I, but not type III colla-gen and DSP (D'Souza et al., 1995). Our knowledge of gene,transcription factor, and growth factor expression during den-tal injury is still in its infancy but will contribute significantly toour understanding of the molecular control of odontoblast-likecell differentiation and repair.

(III-2) IN VIVO EXPERIMENTAL MODELSOF DENTAL INJURY AND REPAIR

Preparation of class V cavities in sound human teeth extractedfor orthodontic reasons has been widely used for the assess-ment of restorative procedures and materials. Despite the limi-tations of such a model, where the dentin thickness is limitedand pathologic changes in the dentin resulting from the cariesare absent, it probably represents the closest to the real clinicalsituation. Ethical considerations now prevent the use of suchmodels, with the possible exception of re-analysis of archivalhistological material prepared some years ago.

In vivo animal models provide an acceptable alternative tohuman models, although species differences should be takeninto consideration in the interpretation of tissue responses. Theanimals used include the monkey (Cox et al., 1992), the dog(Tziafas et al., 1998), the ferret (Smith et al., 1990), and the rat(Six et al., 2000). While such approaches are valuable for theexamination of tissue responses in more physiological and clin-ical contexts, the complexity of the model and the control ofpossible experimental variables are important considerations.The surgical procedures giving rise to the tissue injury proba-bly represent the most critical variable and have been alludedto in the preceding section of this review. Cavity preparation iswell-recognized to be technique-sensitive, leading to a varietyof tissue responses, and subsequent cavity treatment andrestoration can also invoke pulpal responses (Mjör, 2002).

Both ethical and cost considerations have favored use ofthe rat model for in vivo studies by many researchers, despitethe small size of the teeth. The special nature of this particularanimal model warrants more detailed comments to put its useinto perspective. The rat pulp demonstrates exceptionalresilience and self-reparative capacity, which must be takeninto account in the interpretation of experimental results. Thecontinuously growing nature of the rat incisor limits its useful-ness, and access to the mandibular first molar is difficult dur-ing surgery. The maxillary molar provides better surgicalaccess, and Ohshima (1990) proposed cavity preparation on themesial aspect of these teeth. Elimination of the gingival margin

by electrosurgery allows cervical cavities to be prepared in azone facing the middle of the coronal pulp in this tooth, there-by avoiding preparations near the tip of the pulp horn, whereexcessive reactionary dentinogenesis can be a problem(Goldberg et al., 1999). Cavities should be prepared with a tung-sten carbide bur within 2 sec, followed by a short rinse withwater, to avoid heat damage. The dorsal surgical presentationof the animal precludes use of water cooling during cutting;otherwise, water inhalation is a risk. The cavity is calibrated,and its size (width and depth) largely depends on the size ofthe bur.

The expertise of the investigator is crucial in these surgicalprocedures, and this reinforces the view that cavity preparationis a critical variable (Mjör, 2002). After cavity preparation, pulpexposure can be performed by mechanical perforation of thecavity floor with a steel probe. This approach avoids the exten-sive pulp damage caused by exposure during cutting with thebur, but does lead to dentin fragments being pushed into thepulp. While this tissue debris does not appear to invoke aninflammatory response (Decup et al., 2000), auto-induction ofreparative dentinogenesis can be seen on the surfaces of thedentin particles (Tziafas et al., 1992). Even in the absence of anypulp-capping agent, spontaneous calcospherite formation canbe observed in the mesial area of the coronal pulp chamberafter exposed cavity preparation in the rat. This leads to depo-sition of an osteodentin-like matrix interspersed with unminer-alized pulp remnants. Thus, any evaluation of the biologicalproperties of implanted molecules must be compared withappropriate controls. Implanted molecules may be appliedbound to a carrier (collagen) or adsorbed onto the surfaces ofagarose beads or other substrates prior to the sealing of the cav-ity with a glass-ionomer cement to avoid bacterial infection.Generally, inflammatory processes will have subsided within 8days, during which progenitor cell recruitment for reparativedentinogenesis will have occurred. Reparative dentinogenesisis initiated within 2 wks, and in the presence of calciumhydroxide, a thick dentin bridge will form within the subse-quent 2 wks. While this rat model can be capricious in thehands of an inexperienced investigator, it is capable of provid-ing valuable data in more experienced hands. Use of alterna-tive, higher species of animals is not always viable in manycountries because of ethical restrictions, or due to a high cost.

(III-3) IN VITRO MODELSOF DENTAL INJURY AND REPAIR

In vitro culture models have found widespread use because oftheir apparent simplicity. Cells emerging from cultured pulpexplants give rise to a population of pulp-like cells with specif-ic characteristics (Nakashima, 1991; Kasugai et al., 1993; Hankset al., 1998; Ueno et al., 2001). After 15-20 days of culture, theyare strongly positive for alkaline phosphatase and form miner-alization nodules that stain positively with the von Kossa reac-tion. The nodules have been shown to be calcium- and phos-phorus-rich by electron probe analyses (Stanislawski et al.,1997). Once differentiated, these pulp cells display an �-smoothmuscle actin-rich phenotype and consequently may be derivedfrom myofibroblasts or pericytes (Alliot-Licht et al., 2001).Pericytes have also been reported to be possible osteoblastprogenitor cells (Brighton et al., 1992). What is unclear is theextent to which cell selection occurs during pulp explant cul-ture. Both the culture conditions and outgrowth of cells fromthe explant will almost certainly favor selection of particular




populations of migratory cells. Caution is therefore required inextrapolation of data from such cell cultures to the pulp in gen-eral.

Pulp cells in culture express collagen types I and III,osteonectin, osteopontin, fibronectin, and alkaline phos-phatase, but not DPP or DSP. Selected clones of cells, however,express DSP and other molecules considered as odontoblast-specific markers, suggesting that spontaneous transformationof cell phenotype can occur during culture (Hanks et al., 1998).In some conditions, such as addition of �-glycerophosphate,cultured pulp cells will form mineralized nodules and expressodontoblast markers (About et al., 2000).

In addition to primary pulp cell cultures, attempts havebeen made to derive immortalized odontoblast-like cell linesfrom dental papilla cells. Transfection with simian virus 40 largeT antigen gave rise to a cell line that was positive for DSPP, typeI collagen, and alkaline phosphatase (MacDougall et al., 1995).Cell lines that expressed collagen type I, alkaline phosphatase,and osteocalcin have been reported after immortalization withhuman papillomavirus 18 (HPV 18 E6/E7) (Thonemann andSchmalz, 2000). With use of a gene for telomerase, immortalizedcells possessing an odontoblast-like phenotype were alsoobtained. These cells express DMP-1 and DSPP and produce amineralized dentin matrix both in vivo and in vitro (Hao et al.,2002). These immortalized odontoblast-like cell lines may bevaluable tools for molecular investigations, but their ability toundergo cell division is at variance with the post-mitotic natureof odontoblasts under physiological conditions. The signifi-cance of these cell cycle differences remains to be established.

Tooth organ culture has been adopted for the study ofdentinogenic events in rat incisor teeth, and this model afford-ed the important observation that separation of odontoblastsfrom contact with their dentin matrix leads to marked changesin their morphology (Heywood and Appleton, 1984). Althoughodontoblasts continue to show secretory activity when grown inassociation with the dentin matrix after pulp extirpation(Embery and Smalley, 1980), their functional behavior can prob-ably be studied for time periods of only fewer than 24 hrs beforecellular atrophy becomes significant. It is possible to grow thesetissues for periods of up to 2 to 4 wks if thick slices of human orrat teeth are grown intact in culture without removal of the pulptissues (Magloire et al., 1995; Sloan et al., 1998; Dobie et al., 2002).This approach also has the advantage that normal architecturalrelationships of the dentin-pulp complex are maintained duringculture. While such models are valuable for studying eventsover periods of some days (Sloan and Smith, 1999), cautionmust be exercised over longer periods of weeks in the absenceof information on whether or not physiological cell behavior ismaintained. Nevertheless, such organ culture models providevaluable alternatives when in vivo experimentation is inappro-priate and close control of experimental variables is required.

(III-4) CALCIUM HYDROXIDE AS A REFERENCEFOR INDUCTION OF REPARATIVE DENTINOGENESIS

Calcium hydroxide has been widely used as a pulp-cappingagent for more than 60 years (Zander, 1939) and has been per-ceived by some as the gold standard against which other cap-ping procedures should be evaluated. Its use on the exposedpulp can induce a reparative dentinogenic response leading todentin bridge formation (Fig. 3). However, Ca(OH)2 does nothave a specific target of action, and its behavior will be reviewedto assess the validity of use of this material as a reference.

As a pulp-capping agent, the strongly alkaline pH ofCa(OH)2 may contribute significantly to its action. Initially, thishigh pH appears to cause local necrosis of the pulp tissuearound the injury site, and migration of pulp cells to this areais seen after approximately 2 wks (Schroder, 1985). While highpH environments are not generally conducive to biologicalactivity, in this situation, Ca(OH)2 may prevent bacterial infec-tion, thereby dampening inflammatory responses and provid-ing a relatively sterile environment in which subsequent repaircan occur. As cells migrate into the necrotic area, odontoblast-like cell differentiation commences and a mineralized repara-tive dentin bridge is formed (Schroder, 1985; Mjör et al., 1991).Two important questions arise from these observations: (1)What is the nature of the recruited progenitor cells for dentinbridge formation? and (2) What signaling processes are respon-sible for induction of differentiation of these cells to odonto-blast-like cells?

DNA synthesis can be inhibited in the presence of Ca(OH)2in vitro. Alkaline phosphatase activity and protein synthesis arediminished, although incorporation of the amino-acids leucineand proline, precursors of collagen and non-collagenous pro-teins, is increased (Alliot-Licht et al., 1994). Human dental pulpcells cultured on Ca(OH)2 differentiate into odontoblast-likecells after 4 wks, synthesize type I but not type III collagen, anddisplay apical accumulation of actin and vimentin (Seux et al.,1991). Pulp cell activities observed after Ca(OH)2 applicationare dependent on the stage of examination and the tissue con-ditions prevailing at that time.

The signaling processes responsible for Ca(OH)2-inducedodontoblast-like cell differentiation remain elusive. To suggestthat differentiation represents spontaneous differentiationimplies activation of an endogenous repair mechanism. Such aconcept may prove founded if Ca(OH)2 is able to solubilize bio-active molecules with appropriate signaling functions from thedentin matrix (Tziafas et al., 2000). It has also been suggestedthat alterations in calcium levels in the cellular environmentcan invoke responses of apoptosis or differentiation of odonto-blast-like cells (Kardos et al., 1998). However, there are proba-bly aberrations in signaling, as suggested by the rather abnor-mal appearance of the initial matrix of the dentin bridge, whichis usually of the osteodentin type rather than true tubular orth-odentin. Discontinuities in dentin bridge formation give rise toso-called "tunnel defects" (Cox et al., 1996), the presence ofwhich impairs the bridge's properties as a permeability barrier.During later bridge formation, the matrix structure oftenbecomes more regular, resembling true tubular orthodentin.

Thus, Ca(OH)2 appears to have relatively non-specificeffects as an inducer of reparative dentinogenesis and consid-erable heterogeneity in its action. However, the broad experi-ence gained with Ca(OH)2 in a variety of experimental andclinical situations makes it a useful treatment for comparativepurposes for the evaluation of new pulp-capping agents.

(III-5) SIGNALING MOLECULES FOR PULP REPAIR

(III-5-1) Diversity of molecules and experimentalapproaches

The possibility for the use of a variety of biological macromol-ecules for signaling repair in pulp-capping and other treatmentsituations has offered exciting opportunities for the develop-ment of novel biomimetic approaches in restorative dentistry.A plethora of molecules—including growth factors, dentin





matrix proteins, and extracellularmatrix molecules—has been evaluatedfor their potential contributions to den-tal tissue repair. These studies werelargely derived from the observationsthat dentin matrix is auto-inductive,obviating the requirement for dentalepithelium to signal odontoblast-likecell differentiation. The histopathologicobservations of dentinogenesis on thesurfaces of dentin chips pushed into thepulp during cavity preparation led tostudies which showed reparativedentinogenesis when demineralizeddentin matrix (Tziafas and Kolokuris,1990) or soluble dentin matrix extracts(Smith et al., 1990) were implanted intoexposed pulps. These observations cat-alyzed more detailed studies of specificmatrix molecules—such as fibronectin(Tziafas et al., 1992), bone sialoprotein(Decup et al., 2000), and the amelogeninpolypeptides A+4 and A-4 (Six et al.,2002c)—for the evaluation of theirdentinogenic effects during repair. Thepresence of growth factors in dentinmatrix and their putative role in signal-ing odontoblast differentiation duringembryogenesis have also led to exami-nation of the effects of TGF-�s, BMPs,FGFs, and IGFs in reparative dentino-genesis (Rutherford et al., 1993, 1994;Nakashima, 1994a,b; Nakashima et al.,1994; Tziafas et al., 1998; Sloan andSmith, 1999). In all of these variousstudies, reparative responses have beenobserved ranging from initial secretionof relatively non-specific atubular fibro-dentin to apparently more specifictubular dentinogenic responses, often superimposed on atubu-lar fibrodentin-like matrix. This raises many questions: Whatconstitutes a specific dentinogenic response as opposed tosecretion of a non-specific mineralized mass? To what degreeare these molecules directly signaling odontoblast-like cell dif-ferentiation or indirectly influencing the tissue environment tofacilitate signaling by other endogenous matrix molecules?Thus, several questions remain as to the signaling mechanismsin odontoblast-like cell differentiation during reparativedentinogenesis and which specific molecules are involved.From the viewpoint of potential clinical applications, suchquestions may be of lesser importance, since the final outcomeof the treatment is the main goal. However, a mechanisticunderstanding of how that outcome is reached is important ifit is to be achieved in a well-controlled manner.

(III-5-2) Strategies for pulp repair using specific classes of molecules

There is a plethora of biological molecules that could potential-ly influence the induction of reparative dentinogenesis andpulp repair. Strategies have been developed to investigategroups of molecules potentially available within the pulp afterinjury for natural mechanisms of repair and/or which may be

able to recapitulate developmental events. A few of thesestrategies will be considered to provide guidance for futurestudies.

Members of the TGF-� family have been popular candi-dates for the induction of reparative dentinogenesis in view oftheir implication in developmental odontoblast differentiation(for review, see Smith and Lesot, 2001). BMP-7 or OP-1 was oneof the first growth factors to be examined in pulp repair. In themonkey, BMP-7/OP1 induced widespread osteodentin forma-tion throughout the pulp, including the total occlusion of theroots of treated teeth (Rutherford et al., 1993, 1994). An irregu-lar reparative osteodentin was deposited in the coronal pulp ofrat molars 30 days after BMP-7 treatment, and extensive repar-ative dentin was observed in the roots of these teeth, beneath acalcio-traumatic line, with complete occlusion of the mesialroot canal in many cases (Six et al., 2002a). These observationshighlight an important aspect of reparative dentinogenesis,namely, that the coronal and root regions of the pulp may pro-vide different environments for repair. The reasons for thesedifferences are unclear, but their elucidation could prove fruit-ful for the development of novel endodontic therapies for clin-ical use. The above studies also indicate that considerationmust be given to the regulation of reparative processes.


Figure 3. In the rat molar model, 8 days after implantation, Ca(OH)2 induces the formation ofa reparative dentinal bridge. P, pulp; C, cavity. 120x.

Figure 4. Thirty days after implantation, BSP induces the formation of a homogeneous mineral-ized tissue (a,b) that totally occludes the pulpal exposure (star). 360x.

Figure 5. MEPE implantation. After 30 days, the mesial part of the pulp (P) and mesial rootcanal were filled with reparative dentin (arrowheads). C, cavity. G, gingiva. 10x.

Figure 6. Structure of the reparative dentin induced by MEPE implantation. Dentin debris andremnants of agarose beads, used as carriers (arrow), are embedded in the reparative dentinthat occludes the perforation. 360x.




Uncontrolled reparative dentinogenesis poses a problem in anendodontic situation, unless apical closure is adequate to pre-vent the spread of mineralization into periapical areas. Controlmight be exerted through the dose-dependency of the repara-tive events, but this requires further investigation. Such issuesunderline the need for a full understanding of the mechanismsof action of molecules such as BMP, before they can be effec-tively exploited for clinical use. BMP-7 can induce new boneformation at ectopic sites, and BMP receptors have been identi-fied within dental pulp (Takeda et al., 1994a,b; Toyono et al.,1997). However, we have little understanding of the identitiesand phenotypes of the targeted cells or of the cellular process-es affected during BMP-induced pulp repair.

In view of the auto-inductive effects of displaced dentinparticles on reparative dentinogenesis in the pulp, the effects ofsome dentin matrix molecules have been examined.Phosphorylated proteins provide potential candidates in thiscontext, and BSP has been selected for several reasons. Thepolyglutamic acid sequences in BSP provide hydroxyapatite(HAP)-binding sites and have the potential to mediate initialnucleation of HAP crystals. Fibrillogenesis of collagen isenhanced by BSP, which is also expressed by odontoblasts butnot by pulp cells (Decup et al., 2000). Implantation of BSP intorat molar exposed pulps led to cellular changes in the pulpwithin 1 wk, and reparative dentinogenesis commenced after 2wks, often in close association with the surface of dentin debrispushed into the pulp during perforation. After 1 mo, the mesialaspect of the coronal pulp was filled with a homogeneous massof atubular dentin (Figs. 4a, 4b). In contrast to the studies withBMP-7, no effect of BSP was observed on the radicular pulp.Thus, these 2 bioactive molecules appear to target differentareas of the pulp for repair. BSP might find clinical applicationin the induction of pulp mineralization in the coronal pulp,whereas BMP-7 could induce occlusion of both the radicularand coronal regions of the pulp (Six et al., 2002b). In the situa-tion of coronal pulp repair, restoration of the physiologicalstructural integrity of the tissue, i.e., a tubular orthodentinmatrix, may be considered preferable, but in an endodonticapplication, apical sealing of the root canal is important, and aless permeable and homogeneous reparative tissue would befavored.

The effects of a parent molecule of the SIBLING familyhave also been investigated. MEPE (matrix extracellular phos-phoglycoprotein), recently identified in odontoblasts(MacDougall et al., 2002), has been implanted via agarose beadsinto rat maxillary molars. After 1 mo, extensive reparativedentin formation was seen in the mesial third of the pulp, withocclusion of the root canal (Figs. 5, 6).

The role of epithelially derived molecular signaling inodontoblast differentiation during embryogenesis is well-established and has led to the examination of these moleculesin pulp repair. A+4 and A-4 are found in dentin and representa low-molecular-mass amelogenin polypeptide (Veis et al.,2000). Their cDNA has been identified in the odontoblastcDNA library and represents an alternatively spliced geneproduct. The first polypeptide is expressed by all the amelo-genin gene exons (A+4), whereas A-4 is expressed by all theexons except the 4th (Veis et al., 2000). Implantation of A+4 andA-4 via agarose beads into rat molar pulps resulted in recruit-ment of a dense layer of cells on the bead surface after 1 wk,suggesting that these molecules have a chemotactic effect (Sixet al., 2002c). In the root, instead of a single layer of odonto-

blasts at the border of the pulp, a double layer was seen, sug-gesting an unusual pattern of induction of odontoblast-like celldifferentiation. After 15 days' implantation of A+4, the reactionwas restricted to an area near the exposure, where a thickdentin bridge developed after 30 days. In contrast, the initialreaction was more diffuse with A-4. Mineralization wasobserved both in the mesial coronal pulp and in the mesial root,and in many instances the latter was totally occluded with adense matrix. Clearly, there is a need to screen a wide variety ofbio-active molecules for potential use in pulp repair and toexamine their mechanisms of action closely.

(IV) ConclusionsThe last decade has proved to be an exciting time for pulp biol-ogy and has led to rapid advances in our knowledge of repairin this tissue. At the start of a new millennium, the use of bio-logical molecules for the development of novel restorativetreatment modalities in clinical dentistry is in sight. Theseapproaches have potential applications in unexposed cavitypreparations for protection of the pulp from harmful effects ofdental materials by increasing the residual dentin thicknessthrough reactionary dentinogenesis, as well as in exposed pulpsituations for restoration of the structural integrity of the dentinwall by reparative dentinogenesis. In the severely compro-mised pulp, it may even be possible to use biological approach-es in endodontic therapy to seal the root canal.

A thorough understanding of the various biologicalprocesses involved in repair will be pivotal to our future suc-cess in developing these approaches. Our review of this areahas highlighted that, despite the considerable progressachieved, there are still numerous fundamental questions to beaddressed. Emerging technologies in molecular and cell biolo-gy will be of crucial importance to our understanding of thecells responsible for repair and the molecular signals that directand regulate these cells. Of fundamental importance will be anunderstanding of the nature and phenotype of the cellsinvolved in repair, their molecular regulation, and the secreto-ry behavior which gives rise to various mineralized matrices.Such knowledge will also underpin progress in tissue engi-neering in this area.

AcknowledgmentsWe acknowledge Dr. Ngampis Six' contributions as a PhD student involved

in the research on BSP, BMP-7, A+4 and A-4, and MEPE. We thank Dr.

Erdjan Salih (Boston), Dr. Bruce Rutherford (Ann Arbor), Dr. Arthur Veis

(Chicago), Dr. Pam DenBesten (San Francisco), and Acologix Inc. for their

kind collaboration. We acknowledge the Université Paris V for an "Axe

d'Excellence" grant, for the Research Grant CNRS-INSERM "Ingénierie tis-

sulaire", the Institut Français pour la Recherche Odontologique-IFRO,

research grants from the Wellcome Trust and UK MRC, and EPSRC and the

European Program COST Odontogenesis for their support.

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Critical Reviews in Oral Biology & Medicine ... · 15(1):13-27 (2004) Crit Rev Oral Biol Med 13 Introduction Dentinogenesis has been studied extensively as investiga- tors attempt